Phospholipases, Nucleic Acids Encoding Them and Methods for Making and Using Them

ABSTRACT

The invention provides novel polypeptides having phospholipase activity, including, e.g., phospholipase A, B, C and D activity, patatin activity, phosphatidic acid phosphatases (PAP)) and/or lipid acyl hydrolase (LAH) activity, nucleic acids encoding them and antibodies that bind to them. Industrial methods, e.g., oil degumming, and products comprising use of these phospholipases are also provided.

FIELD OF THE INVENTION

This invention relates generally to phospholipase enzymes,polynucleotides encoding the enzymes, methods of making and using thesepolynucleotides and polypeptides. In particular, the invention providesnovel polypeptides having phospholipase activity, nucleic acids encodingthem and antibodies that bind to them. Industrial methods and productscomprising use of these phospholipases are also provided.

BACKGROUND

Phospholipases are enzymes that hydrolyze the ester bonds ofphospholipids. Corresponding to their importance in the metabolism ofphospholipids, these enzymes are widespread among prokaryotes andeukaryotes. The phospholipases affect the metabolism, construction andreorganization of biological membranes and are involved in signalcascades. Several types of phospholipases are known which differ intheir specificity according to the position of the bond attacked in thephospholipid molecule. Phospholipase A1 (PLA1) removes the 1-positionfatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid.Phospholipase A2 (PLA2) removes the 2-position fatty acid to producefree fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes canbe intra- or extra-cellular, membrane-bound or soluble. IntracellularPLA2 is found in almost every mammalian cell. Phospholipase C (PLC)removes the phosphate moiety to produce 1,2 diacylglycerol and phosphateester. Phospholipase D (PLD) produces 1,2-diacylglycerophosphate andbase group. PLC and PLD are important in cell function and signaling.PLD had been the dominant phospholipase in biocatalysis (see, e.g.,Godfrey, T. and West S. (1996) Industrial enzymology, 299-300, StocktonPress, New York). Patatins are another type of phospholipase, thought towork as a PLA (see for example, Hirschberg H J, et al., (2001), Eur JBiochem 268(19):5037-44).

Common oilseeds, such as soybeans, rapeseed, sunflower seeds, rice branoil, sesame and peanuts are used as sources of oils and feedstock. Inthe oil extraction process, the seeds are mechanically and thermallytreated. The oil is separated and divided from the meal by a solvent.Using distillation, the solvent is then separated from the oil andrecovered. The oil is “degummed” and refined. The solvent content in themeal can be evaporated by thermal treatment in a “desolventizertoaster,” followed by meal drying and cooling. After a solvent had beenseparated by distillation, the produced raw oil is processed into edibleoil, using special degunming procedures and physical refining. It canalso be utilized as feedstock for the production of fatty acids andmethyl ester. The meal can be used for animal rations.

Degumming is the first step in vegetable oil refining and it is designedto remove contaminating phosphatides that are extracted with the oil butinterfere with the subsequent oil processing. These phosphatides aresoluble in the vegetable oil only in an anhydrous form and can beprecipitated and removed if they are simply hydrated. Hydration isusually accomplished by mixing a small proportion of water continuouslywith substantially dry oil. Typically, the amount of water is 75% of thephosphatides content, which is typically 1 to 1.5%. The temperature isnot highly critical, although separation of the hydrated gums is betterwhen the viscosity of the oil is reduced at 50° C. to 80° C.

Many methods for oil degumming are currently used. The process of oildegumming can be enzymatically assisted by using phospholipase enzymes.Phospholipases A1 and A2 have been used for oil degumming in variouscommercial processes, e.g., “ENZYMAX™ degumming” (Lurgi Life ScienceTechnologies GmbH, Germany). Phospholipase C (PLC) also has beenconsidered for oil degumming because the phosphate moiety generated byits action on phospholipids is very water soluble and easy to remove andthe diglyceride would stay with the oil and reduce losses; see e.g.,Godfrey, T. and West S. (1996) Industrial Enzymology, pp. 299-300,Stockton Press, New York; Dahlke (1998) “An enzymatic process for thephysical refining of seed oils,” Chem. Eng. Technol. 21:278-281; Clausen(2001) “Enzymatic oil degumming by a novel microbial phospholipase,”Eur. J. Lipid Sci. Technol. 103:333-340.

High phosphatide oils such as soy, canola and sunflower are processeddifferently than other oils such as palm. Unlike the steam or “physicalrefining” process for low phosphatide oils, these high phosphorus oilsrequire special chemical and mechanical treatments to remove thephosphorus-containing phospholipids. These oils are typically refinedchemically in a process that entails neutralizing the free fatty acidsto form soap and an insoluble gum fraction. The neutralization processis highly effective in removing free fatty acids and phospholipids butthis process also results in significant yield losses and sacrifices inquality. In some cases, the high phosphatide crude oil is degummed in astep preceding caustic neutralization. This is the case for soy oilutilized for lecithin wherein the oil is first water or acid degummed.

Phytosterols (plant sterols) are members of the “triterpene” family ofnatural products, which includes more than 100 different phytosterolsand more than 4000 other types of triterpenes. In general, phytosterolsare thought to stabilize plant membranes, with an increase in thesterol/phospholipid ration leading to membrane rigidification.Chemically, phytosterols closely resemble cholesterol in structure andare thought to regulate membrane fluidity in plant membranes, as doescholesterol in animal membranes. The major phytosterols areβ-sitosterol, campesterol and stigmasterol. Others include stigmastanol(β-sitostanol), sitostanol, desmosterol, dihydrobrassicasterol,chalinasterol, poriferasterol, clionasterol and brassicasterol.

Plant sterols are important agricultural products for health andnutritional industries. They are useful emulsifiers for cosmeticmanufacturers and supply the majority of steroidal intermediates andprecursors for the production of hormone pharmaceuticals. The saturatedanalogs of phytosterols and their esters have been suggested aseffective cholesterol-lowering agents with cardiologic health benefits.Plant sterols reduce serum cholesterol levels by inhibiting cholesterolabsorption in the intestinal lumen and have immunomodulating propertiesat extremely low concentrations, including enhanced cellular response ofT lymphocytes and cytotoxic ability of natural killer cells against acancer cell line. In addition, their therapeutic effect has beendemonstrated in clinical studies for treatment of pulmonarytuberculosis, rheumatoid arthritis, management of HIV-infested patientsand inhibition of immune stress in marathon runners.

Plant sterol esters, also referred to as phytosterol esters, wereapproved as GRAS (Generally Recognized As Safe) by the US Food and DrugAdministration (FDA) for use in margarines and spreads in 1999. InSeptember 2000, the FDA also issued an interim rule that allowshealth-claims labeling of foods containing phytosterol ester.Consequently enrichment of foods with phytosterol esters is highlydesired for consumer acceptance.

Soybean oil is widely used and is an important foodstuff, accounting for˜30% of the oil production from seeds and fruits. Soybeans contain only20% oil, and the extraction is usually done by using a solvent such ashexane on a commercial scale. The recognized quality of its oil and thenutritive value of the meal protein make soya bean a primary oilseed.Before extraction, soybeans must be cleaned, cracked and flaked asefficient solvent extraction of oil requires that every oil cell isbroken to improve the mass transfer. Cell walls mostly composed ofcellulose, associated with hemicelluloses, pectic substances andlignin), could also be broken by means of enzymes, to achieve asignificant improvement in extraction yields and rates.

Diacylglycerol (DAG) oil is an edible oil containing 80% or greateramount of DAG than natural fatty acids. It has been shown in humans thatpostprandial elevation of triglyceride in chylomicrons is markedlysmaller after ingestion of a DAG oil emulsion compared to a TAG oil witha similar fatty acid composition. In studies using Japanese men andAmerican men and women, long-term DAG oil consumption promoted weightloss and body fat reduction. One study showed that substitution of DAGoil for ordinary cooking oil reduces the incidence of obesity and otherrisk factors.

SUMMARY OF THE INVENTION

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171 or SEQ ID NO:173, over a region of at least about 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400,2450, 2500, or more residues, and in one aspect the nucleic acid encodesat least one polypeptide having a phospholipase (PL) activity, e.g., aphospholipase A, C or D activity, or any combination of phospholipaseactivity, for example, a PL A, PL C and/or PL D activity—as amultifunctional activity. In one aspect, the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, ormore, or complete (100%) sequence identity to SEQ ID NO:1 over a regionof at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 moreconsecutive residues, and in one aspect the nucleic acid encodes atleast one polypeptide having a phospholipase (PL) activity, e.g., aphospholipase A, C or D activity, or any combination of phospholipaseactivity, for example, a PLA, PL C and/or PL D activity—as amultifunctional activity. In one aspect, the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:3over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850 or more residues, and in one aspect the nucleic acid encodes atleast one polypeptide having a phospholipase (PL) activity, e.g., aphospholipase A, C or D activity, or any combination of phospholipaseactivity, for example, a PL A, PL C and/or PL D activity—as amultifunctional activity. In one aspect, the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:5over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850 or more residues, and in one aspect the nucleic acid encodes atleast one polypeptide having a phospholipase (PL) activity, e.g., aphospholipase A, C or D activity, or any combination of phospholipaseactivity, for example, a PLA, PL C and/or PL D activity—as amultifunctional activity. In one aspect, the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:7over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850 or more residues, and in one aspect the nucleic acid encodes atleast one polypeptide having a phospholipase (PL) activity, e.g., aphospholipase A, C or D activity, or any combination of phospholipaseactivity, for example, a PL A, PL C and/or PL D activity—as amultifunctional activity. In one aspect, the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

In alternative aspects, the isolated or recombinant nucleic acid encodesa polypeptide comprising a sequence as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO: 82, SEQ IDNO:84, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ IDNO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ IDNO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,SEQ ID NO:172, or SEQ ID NO:174. In one aspect these polypeptides have aphospholipase, e.g., a phospholipase A, B, C or D activity, or anycombination of phospholipase activity, for example, a PLA, PL C and/orPL D activity—as a multifunctional activity.

In one aspect, the sequence comparison algorithm is a BLAST algorithm,such as a BLAST version 2.2.2 algorithm. In one aspect, the filteringsetting is set to blastall-p blastp-d “nr pataa”-F F and all otheroptions are set to default.

In one aspect, the phospholipase activity comprises catalyzinghydrolysis of a glycerolphosphate ester linkage (i.e., cleavage ofglycerolphosphate ester linkages). The phospholipase activity cancomprise catalyzing hydrolysis of an ester linkage in a phospholipid ina vegetable oil. The vegetable oil phospholipid can comprise an oilseedphospholipid. The phospholipase activity can comprise a phospholipase C(PLC) activity; a phospholipase A (PLA) activity, such as aphospholipase A1 or phospholipase A2 activity; a phospholipase D (PLD)activity, such as a phospholipase D1 or a phospholipase D2 activity; aphospholipase B (PLB) activity, e.g., a phospholipase and alysophospholipase (LPL) activity or a phospholipase and alysophospholipase-transacylase (LPTA) activity or a phospholipase and alysophospholipase (LPL) activity and lysophospholipase-transacylase(LPTA) activity; or patatin activity, or a combination thereof. Thephospholipase activity can comprise hydrolysis of a glycoprotein, e.g.,as a glycoprotein found in a potato tuber. The phospholipase activitycan comprise a patatin enzymatic activity. The phospholipase activitycan comprise a lipid acyl hydrolase (LAH) activity. In one aspect, aphospholipase of the invention can have multifunctional activity, e.g.,a combination of one or more of the enzyme activities described herein,for example, a phospholipase of the invention can have PLC and PLAactivity; PLB and PLA activity; PLC and PLD activity; PLC and PLBactivity; PLB and patatin activity; PLC and patatin activity; PLD andPLA; PLD, PLA, PLB and PLC activity; or PLD, PLA, PLB, PLC and patatinactivity; or, a phospholipase and a lysophospholipase (LPL) activity ora phospholipase and a lysophospholipase-transacylase (LPTA) activity ora phospholipase and a lysophospholipase (LPL) activity andlysophospholipase-transacylase (LPTA) activity, or any combinationthereof.

For example, in one aspect, a polypeptide of the invention isenzymatically active, but lacks a lipase activity, e.g., lacks anyenzymatic activity that affects a neutral oil (triglyceride) fraction.It may be desirable to use such a polypeptide in a particular process,e.g., in a degumming process where it is important that the neutral oilfraction not be harmed (diminished, e.g., hydrolyzed). Thus, in oneaspect, the invention provides a degumming process comprising use of apolypeptide of the invention having a phospholipase activity, but not alipase activity.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a phospholipase activity which is thermostable. Thepolypeptide can retain a phospholipase activity under conditionscomprising a temperature range of between about 20° C. to about 30° C.,between about 25° C. to about 40° C., between about 37° C. to about 95°C.; between about 55° C. to about 85° C., between about 70° C. to about95° C., or, between about 90° C. to about 95° C. In another aspect, theisolated or recombinant nucleic acid encodes a polypeptide having aphospholipase activity which is thermotolerant. The polypeptide canretain a phospholipase activity after exposure to a temperature in therange from greater than 37° C. to about 95° C. or anywhere in the rangefrom greater than 55° C. to about 85° C. In one aspect, the polypeptideretains a phospholipase activity after exposure to a temperature in therange from greater than 90° C. to about 95° C. at pH 4.5.

The polypeptide can retain a phospholipase activity under conditionscomprising about pH 8, pH 7.5, pH 7, pH 6.5, pH 6.0, pH 5.5, pH 5, or pH4.5. The polypeptide can retain a phospholipase activity underconditions comprising a temperature range of between about 40° C. toabout 70° C.

In one aspect, the isolated or recombinant nucleic acid comprises asequence that hybridizes under stringent conditions to a sequence as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 or SEQ ID NO:173,wherein the nucleic acid encodes a polypeptide having a phospholipaseactivity. The nucleic acid can at least about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850 or residues in length or the full length of the geneor transcript, with or without a signal sequence, as described herein.The stringent conditions can be highly stringent, moderately stringentor of low stringency, as described herein. The stringent conditions caninclude a wash step, e.g., a wash step comprising a wash in 0.2×SSC at atemperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide with a phospholipase, e.g., a phospholipase,activity, wherein the probe comprises at least 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, or more, consecutive bases of a sequence of theinvention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, or SEQ ID NO:7, and the probe identifies the nucleic acidby binding or hybridization. The probe can comprise an oligonucleotidecomprising at least about 10 to 50, about 20 to 60, about 30 to 70,about 40 to 80, or about 60 to 100 consecutive bases of a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide with a phospholipase, e.g., a phospholipaseactivity, wherein the probe comprises a nucleic acid of the invention,e.g., a nucleic acid having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or complete (100%) sequence identity to SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5 and/or SEQ ID NO:7, or a subsequence thereof, overa region of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 ormore consecutive residues; and, in one aspect, the sequence identitiesare determined by analysis with a sequence comparison algorithm or byvisual inspection.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a phospholipaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more consecutive basesof the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 or more residues of a nucleic acid of the invention, and a secondmember having a sequence as set forth by about the first (the 5′) 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residuesof the complementary strand of the first member.

The invention provides phospholipases generated by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. The invention provides methods of making a phospholipaseby amplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having a phospholipase activity comprising amplification ofa template nucleic acid with an amplification primer sequence paircapable of amplifying a nucleic acid sequence of the invention, orfragments or subsequences thereof. The amplification primer pair can bean amplification primer pair of the invention.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse, a rat, a cow, a sheepor another mammal.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant. Theinvention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a corn seed, a wheat kernel, an oilseed, arapeseed (a canola plant), a soybean seed, a palm kernel, a sunflowerseed, a sesame seed, a peanut, rice or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a phospholipase message in acell comprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a phospholipase message in acell comprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention. The antisense oligonucleotide can bebetween about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80,about 60 to 100, about 70 to 110, or about 80 to 120 bases in length.

The invention provides methods of inhibiting the translation of aphospholipase, e.g., a phospholipase, message in a cell comprisingadministering to the cell or expressing in the cell an antisenseoligonucleotide comprising a nucleic acid sequence complementary to orcapable of hybridizing under stringent conditions to a nucleic acid ofthe invention. The invention provides double-stranded inhibitory RNA(RNAi) molecules comprising a subsequence of a sequence of theinvention. In one aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more duplex nucleotides in length. The inventionprovides methods of inhibiting the expression of a phospholipase, e.g.,a phospholipase, in a cell comprising administering to the cell orexpressing in the cell a double-stranded inhibitory RNA (iRNA), whereinthe RNA comprises a subsequence of a sequence of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, or more, or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention (e.g., SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120 or SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ IDNO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ IDNO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,SEQ ID NO:172, or SEQ ID NO:174) over a region of at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 400, 450, 500, 550 or 600 ormore residues, or over the full length of the polypeptide; and, in oneaspect, the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

In one aspect, the invention provides an isolated or recombinantpolypeptide comprising an amino acid sequence having at least about 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQID NO:2. In one aspect, the invention provides an isolated orrecombinant polypeptide comprising an amino acid sequence having atleast about 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to SEQ ID NO:4. In one aspect, the inventionprovides an isolated or recombinant polypeptide comprising an amino acidsequence having at least about 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to SEQ ID NO:6. In oneaspect, the invention provides an isolated or recombinant polypeptidecomprising an amino acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto SEQ ID NO:8.

The invention provides isolated or recombinant polypeptides encoded by anucleic acid of the invention. In alternative aspects, the polypeptidecan have a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,or SEQ ID NO:174. The polypeptide can have a phospholipase activity,e.g., a phospholipase A, B, C or D activity, or any combination ofphospholipase activity, for example, a PL A, PL C and/or PL Dactivity—as a multifunctional activity. For example, in one aspect, apolypeptide of the invention is enzymatically active, but lacks a lipaseactivity, e.g., lacks any enzymatic activity that affects a neutral oil(triglyceride) fraction. In one aspect, the invention provides adegumming process comprising use of a polypeptide of the inventionhaving a phospholipase activity, but not a lipase activity, such that inthe degumming process any neutral oil fraction is not harmed(diminished, altered, degraded, e.g., hydrolyzed).

The invention provides isolated or recombinant polypeptides comprising apolypeptide of the invention lacking a signal sequence. In one aspect,the polypeptide lacking a signal sequence has at least 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to residues 30 to 287 of SEQ ID NO:2,an amino acid sequence having at least 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to residues 25 to 283 of SEQ IDNO:4, an amino acid sequence having at least 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to residues 26 to 280 of SEQ IDNO:6, or, an amino acid sequence having at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to residues 40 to 330 ofSEQ ID NO:8. The sequence identities can be determined by analysis witha sequence comparison algorithm or by visual inspection.

Another aspect of the invention provides an isolated or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutivebases of a polypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto. The peptide can be, e.g., an immunogenic fragment, a motif(e.g., a binding site) or an active site.

In one aspect, the isolated or recombinant polypeptide of the invention(with or without a signal sequence) has a phospholipase activity. In oneaspect, the phospholipase activity comprises catalyzing hydrolysis of aglycerolphosphate ester linkage (i.e., cleavage of glycerolphosphateester linkages). The phospholipase activity can comprise catalyzinghydrolysis of an ester linkage in a phospholipid in a vegetable oil. Thevegetable oil phospholipid can comprise an oilseed phospholipid. Thephospholipase activity can comprise a phospholipase C (PLC) activity; aphospholipase A (PLA) activity, such as a phospholipase A1 orphospholipase A2 activity; a phospholipase D (PLD) activity, such as aphospholipase D1 or a phospholipase D2 activity; a phospholipase B (PLB)activity, e.g., a phospholipase and a lysophospholipase (LPL) activityor a phospholipase and a lysophospholipase-transacylase (LPTA) activityor a phospholipase and a lysophospholipase (LPL) activity andlysophospholipase-transacylase (LPTA) activity; or patatin activity, ora combination thereof. For example, in one aspect a phospholipasecomprises a combination of one or more of the enzyme activitiesdescribed herein, for example, an phospholipase can have PLC and PLAactivity; PLB and PLA activity; PLC and PLD activity; PLC and PLBactivity; PLB and patatin activity; PLC and patatin activity; PLD andPLA; PLD, PLA, PLB and PLC activity; or PLD, PLA, PLB, PLC and patatinactivity; or, a phospholipase and a lysophospholipase (LPL) activity ora phospholipase and a lysophospholipase-transacylase (LPTA) activity ora phospholipase and a lysophospholipase (LPL) activity andlysophospholipase-transacylase (LPTA) activity, or any combinationthereof.

The phospholipase activity can comprise hydrolysis of a glycoprotein,e.g., as a glycoprotein found in a potato tuber. The phospholipaseactivity can comprise a patatin enzymatic activity. The phospholipaseactivity can comprise a lipid acyl hydrolase (LAH) activity.

In one aspect, the phospholipase activity is thermostable. Thepolypeptide can retain a phospholipase activity under conditionscomprising a temperature range of between about 20 to about 30° C.,between about 25° C. to about 40° C., between about 37° C. to about 95°C., between about 55° C. to about 85° C., between about 70° C. to about95° C., or between about 90° C. to about 95° C. In another aspect, thephospholipase activity can be thermotolerant. The polypeptide can retaina phospholipase activity after exposure to a temperature in the rangefrom greater than 37° C. to about 95° C., or in the range from greaterthan 55° C. to about 85° C. In one aspect, the polypeptide can retain aphospholipase activity after exposure to a temperature in the range fromgreater than 90° C. to about 95° C. at pH 4.5.

In one aspect, the polypeptide can retain a phospholipase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4or less (more acidic). In one aspect, the polypeptide can retain aphospholipase activity under conditions comprising about pH 7, pH 7.5 pH8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more (more basic).

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous phospholipase or non-phospholipase signalsequence.

The invention provides isolated or recombinant peptides comprising anamino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to residues 1 to 29 of SEQ ID NO:2, at least 95%, 96%,97%, 98%, 99%, or more sequence identity to residues 1 to 24 of SEQ IDNO:4, at least 95%, 96%, 97%, 98%, 99%, or more sequence identity toresidues 1 to 25 of SEQ ID NO:6, or at least 95%, 96%, 97%, 98%, 99%, ormore sequence identity to residues 1 to 39 of SEQ ID NO:8, and to othersignal sequences as set forth in the SEQ ID listing, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection. These peptides can act assignal sequences on its endogenous phospholipase, on anotherphospholipase, or a heterologous protein (a non-phospholipase enzyme orother protein). In one aspect, the invention provides chimeric proteinscomprising a first domain comprising a signal sequence of the inventionand at least a second domain. The protein can be a fusion protein. Thesecond domain can comprise an enzyme. The enzyme can be a phospholipase.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP) of the invention or a catalyticdomain (CD), or active site, of a phospholipase of the invention and atleast a second domain comprising a heterologous polypeptide or peptide,wherein the heterologous polypeptide or peptide is not naturallyassociated with the signal peptide (SP) or catalytic domain (CD). In oneaspect, the heterologous polypeptide or peptide is not a phospholipase.The heterologous polypeptide or peptide can be amino terminal to,carboxy terminal to or on both ends of the signal peptide (SP) orcatalytic domain (CD).

The invention provides isolated or recombinant nucleic acids encoding achimeric polypeptide, wherein the chimeric polypeptide comprises atleast a first domain comprising signal peptide (SP) or a catalyticdomain (CD), or active site, of a polypeptide of the invention, and atleast a second domain comprising a heterologous polypeptide or peptide,wherein the heterologous polypeptide or peptide is not naturallyassociated with the signal peptide (SP) or catalytic domain (CD).

In one aspect, the phospholipase activity comprises a specific activityat about 37° C. in the range from about 10 units per milligram to about100 units per milligram of protein. In another aspect, the phospholipaseactivity comprises a specific activity from about 100 units permilligram to about 1000 units per milligram, from about 500 units permilligram to about 750 units per milligram of protein. Alternatively,the phospholipase activity comprises a specific activity at 37° C. inthe range from about 100 to about 500 units per milligram of protein. Inone aspect, the phospholipase activity comprises a specific activity at37° C. in the range from about 500 to about 1200 units per milligram ofprotein. In another aspect, the phospholipase activity comprises aspecific activity at 37° C. in the range from about 750 to about 1000units per milligram of protein. In another aspect, the thermotolerancecomprises retention of at least half of the specific activity of thephospholipase at 37° C. after being heated to the elevated temperature.Alternatively, the thermotolerance can comprise retention of specificactivity at 37° C. in the range from about 500 to about 1200 units permilligram of protein after being heated to the elevated temperature.

The invention provides an isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

The invention provides phospholipase enzymes, and the nucleic acids thatencode them, having a sequence of any of the exemplary phospholipases ofthe invention with one or more or all of the glycosylation sitesaltered, as described above. Thus, the invention provides methods ofmaking variant phospholipase coding sequences having increasedexpression in a host cell, where the method comprises modifying aphospholipase coding sequence of the invention such that one, several orall N-linked glycosylation site coding motifs are modified to anon-glycosylated motif. The invention also provides phospholipase codingsequence made by this process, and the enzymes they encode.

The invention provides methods for making a variant phospholipase codingsequence encoding a phospholipase having increased resistance to aprotease comprising modifying an amino acid equivalent to position 131of SEQ ID NO:2 to one, several or all of the following residues: Lysine(K); Serine (S); Glycine (G); Arginine (R); Glutamine (Q); Alanine (A);Isoleucine (I); Histidine (H); Phenylalanine (F); Threonine (T);Methionine (M) Leucine (L), including variants to SEQ ID NO:2 (and thenucleic acid that encode them) having these exemplary modifications. Theinvention also provides isolated, synthetic or recombinantphospholipases encoded by a sequence made by this method.

The invention provides methods for making a variant phospholipase codingsequence encoding a phospholipase having decreased resistance to aprotease comprising modifying an amino acid equivalent to position 131of SEQ ID NO:2 to one, several or all of the following residues:Tryptophan (W); Glutamate (E); Tyrosine (Y), including variants to SEQID NO:2 (and the nucleic acid that encode them) having these exemplarymodifications. The invention also provides isolated, synthetic orrecombinant phospholipases encoded by a sequence made by this method.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different phospholipase, a different enzyme oranother protein. In one aspect, the second domain can be a polypeptideand the heterodimer can be a fusion protein. In one aspect, the seconddomain can be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having a phospholipaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain (e.g., a fusion protein). In one aspect, a polypeptide of theinvention is immobilized on a cell, a vesicle, a liposome, a film, amembrane, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, crystals,a tablet, a pill, a capsule, a powder, an agglomerate, a surface, aporous structure, an array or a capillary tube. In one aspect, apolypeptide of the invention is immobilized on materials such as grains,husks, bark, skin, hair, enamel, bone, shell and materials deriving fromthem, or animal feed materials, or a combination thereof.

Polypeptides of the invention (e.g., phospholipases) can be also presentalone or as mixture of phospholipases or phospholipases and otherhydrolytic enzymes such as cellulases, xylanases, proteases, lipases,amylases, or redox enzymes such as laccases, peroxidases, catalases,oxidases, or reductases. They can be formulated in a solid form such asa powder, lyophilized preparations, granules, tablets, bars, crystals,capsules, pills, pellets, or in a liquid form such as an aqueoussolution, an aerosol, a gel, a paste, a slurry, an aqueous/oil emulsion,a cream, a capsule, vesicular, or micellar suspension. In one aspect,these formulations of the invention can comprise any or a combination ofthe following ingredients: polyols such as polyethylene glycols,polyvinylalcohols, glycerol, sugars such as sucrose, sorbitol,trehalose, glucose, fructose, maltose, gelling agents such as guar gums,carageenans, alginates, dextrans, cellulosic derivatives, pectins, saltssuch as sodium chloride, sodium sulfate, ammonium sulfate, calciumchloride, magnesium chloride, zinc chloride, zinc sulfate, salts offatty acids and their derivatives, metal chelators such as EDTA, EGTA,sodium citrate, antimicrobial agents such as fatty acids, derivativesthereof, parabens, sorbates, benzoates, additionally compounds to blockthe impact of proteases such as bulk proteins such as BSA, wheathydrolysates, borate compounds, emulsifiers such as non-ionic and ionicdetergents may used alone or in combination, phytosterols, vitamins,amino acids, reducing agents, such as cysteine or antioxidant compoundssuch as ascorbic acid may be included as well dispersants.

In one aspect, cross-linking and protein modification such aspegylation, fatty acid modification and glycosylation are used toimprove the stability of a polypeptide of the invention (e.g., enzymestability). In one aspect, the polyols and/or sugars comprise from about5% to about 60%, or more, of the formulation, from about 10% to about50% of the formulation, from about 20% to about 40% of the formulation,or from about 5% to about 20% of the formulation. In another aspect, thegelling agents comprise from about 0.5% to about 10% of the formulation,from about 1% to about 8% of the formulation, from about 2% to about 5%of the formulation, or from about 0.5% to about 3% of the formulation.In another aspect, the salts such as sodium chloride, sodium sulfate,ammonium sulfate, calcium chloride and/or magnesium chloride comprisefrom about 1% to about 30% of the formulation, from about 2% to about20% of the formulation, from about 5% to about 15% of the formulation,or from about 1% to about 10% of the formulation. In another aspect,zinc chloride is present in the formulation at concentrations comprisingfrom about 0.1 mM to about 20 mM, from about 0.5 mM to about 10 mM, fromabout 1 mM to about 5 mM, or from about 0.1 mM to about 5 mM). In yetanother aspect, zinc sulfate is present in the formulation atconcentrations comprising from about 0.1 mM to about 20 mM, from about0.5 mM to about 10 mM, from about 1 mM to about 5 mM, or from about 0.1mM to about 5 mM). In another aspect, salts of fatty acids and/or theirderivatives comprise from about 5% to about 40% of the formulation, fromabout 10% to about 30% of the formulation, from about 15% to about 25%of the formulation, or from about 5% to about 20% of the formulation. Inanother aspect, metal chelators such as EDTA, EGTA, and/or sodiumcitrate are present in the formulation at concentrations comprising from0.1 mM to about 10 mM), from about 0.5 mM to about 8 mM, from about 1 mMto about 5 mM, or from about 0.1 mM to about 1 mM. In another aspect,antimicrobials such as parabens, sorbates, and/or benzoates comprisefrom about 0.01% to about 10% of the formulation, from about 0.05% toabout 5% of the formulation, from about 0.1% to about 1% of theformulation, or from about 0.05% to about 0.5% of the formulation. Inyet another aspect, bulk proteins such as BSA and/or wheat hydrolysatescomprise from about 1% to about 20% of the formulation, from about 5% toabout 15% of the formulation, from about 2.5% to about 7.5% of theformulation, or from about 1% to about 5% of the formulation. In anotheraspect, emulsifiers such as non-ionic and/or ionic detergents arepresent in the formulation at concentrations comprising from about 1×critical micelle concentration (CMC) to about 10×CMC, from about 2.5×CMCto about 7.5×CMC, from about 1× CMC to about 5×CMC, or from about 3×CMCto about 6×CMC. In another aspect, vitamins, amino acids, reducingagents and/or antioxidant compounds comprise from about 0.1% to about 5%of the formulation, from about 0.5% to about 4% of the formulation, fromabout 1% to about 2.5% of the formulation, or from about 0.1% to about1% of the formulation.

The invention provides arrays comprising an immobilized polypeptide,wherein the polypeptide is a phospholipase of the invention or is apolypeptide encoded by a nucleic acid of the invention. The inventionprovides arrays comprising an immobilized nucleic acid of the invention.The invention provides an array comprising an immobilized antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention.

The invention provides methods of isolating or identifying a polypeptidewith a phospholipase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and, (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying aphospholipase. The invention provides methods of making ananti-phospholipase antibody comprising administering to a non-humananimal a nucleic acid of the invention, or a polypeptide of theinvention, in an amount sufficient to generate a humoral immuneresponse, thereby making an anti-phospholipase antibody.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and, (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. The nucleic acid cancomprise a sequence having at least 85% sequence identity to SEQ ID NO:1over a region of at least about 100 residues, having at least 80%sequence identity to SEQ ID NO:3 over a region of at least about 100residues, having at least 80% sequence identity to SEQ ID NO:5 over aregion of at least about 100 residues, or having at least 70% sequenceidentity to SEQ ID NO:7 over a region of at least about 100 residues,wherein the sequence identities are determined by analysis with asequence comparison algorithm or by visual inspection. The nucleic acidcan comprise a nucleic acid that hybridizes under stringent conditionsto a nucleic acid as set forth in SEQ ID NO:1, or a subsequence thereof;a sequence as set forth in SEQ ID NO:3, or a subsequence thereof; asequence as set forth in SEQ ID NO:5, or a subsequence thereof; or, asequence as set forth in SEQ ID NO:7, or a subsequence thereof. Themethod can further comprise transforming a host cell with the nucleicacid of step (a) followed by expressing the nucleic acid of step (a),thereby producing a recombinant polypeptide in a transformed cell. Themethod can further comprise inserting into a host non-human animal thenucleic acid of step (a) followed by expressing the nucleic acid of step(a), thereby producing a recombinant polypeptide in the host non-humananimal.

The invention provides methods for identifying a polypeptide having aphospholipase activity comprising the following steps: (a) providing apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention, or a fragment or variant thereof, (b) providing aphospholipase substrate; and, (c) contacting the polypeptide or afragment or variant thereof of step (a) with the substrate of step (b)and detecting an increase in the amount of substrate or a decrease inthe amount of reaction product, wherein a decrease in the amount of thesubstrate or an increase in the amount of the reaction product detects apolypeptide having a phospholipase activity. In alternative aspects, thenucleic acid comprises a sequence having at least 85% sequence identityto SEQ ID NO:1 over a region of at least about 100 residues, having atleast 80% sequence identity to SEQ ID NO:3 over a region of at leastabout 100 residues, having at least 80% sequence identity to SEQ ID NO:5over a region of at least about 100 residues, or having at least 70%sequence identity to SEQ ID NO: 7 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection. Inalternative aspects the nucleic acid hybridizes under stringentconditions a sequence as set forth in SEQ ID NO:1, or a subsequencethereof; a sequence as set forth in SEQ ID NO:3, or a subsequencethereof; a sequence as set forth in SEQ ID NO:5, or a subsequencethereof; or, a sequence as set forth in SEQ ID NO:7, or a subsequencethereof.

The invention provides methods for identifying a phospholipase substratecomprising the following steps: (a) providing a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and, (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting an increasein the amount of substrate or a decrease in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of the reaction product identifies the testsubstrate as a phospholipase substrate. In alternative aspects, thenucleic acid can have at least 85% sequence identity to SEQ ID NO:1 overa region of at least about 100 residues, at least 80% sequence identityto SEQ ID NO:3 over a region of at least about 100 residues, at least80% sequence identity to SEQ ID NO: 5 over a region of at least about100 residues, or, at least 70% sequence identity to SEQ ID NO:7 over aregion of at least about 100 residues, wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or byvisual inspection. In alternative aspects, the nucleic acid hybridizesunder stringent conditions to a sequence as set forth in SEQ ID NO:1, ora subsequence thereof; a sequence as set forth in SEQ ID NO:3, or asubsequence thereof; a sequence as set forth in SEQ ID NO:5, or asubsequence thereof; or, a sequence as set forth in SEQ ID NO:7, or asubsequence thereof.

The invention provides methods of determining whether a compoundspecifically binds to a phospholipase comprising the following steps:(a) expressing a nucleic acid or a vector comprising the nucleic acidunder conditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid and vector comprise a nucleic acidor vector of the invention; or, providing a polypeptide of the invention(b) contacting the polypeptide with the test compound; and, (c)determining whether the test compound specifically binds to thepolypeptide, thereby determining that the compound specifically binds tothe phospholipase. In alternative aspects, the nucleic acid sequence hasat least 85% sequence identity to SEQ ID NO:1 over a region of at leastabout 100 residues, at least 80% sequence identity to SEQ ID NO:3 over aregion of at least about 100 residues, least 80% sequence identity toSEQ ID NO:5 over a region of at least about 100 residues, or, at least70% sequence identity to SEQ ID NO:7 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection. Inalternative aspects, the nucleic acid hybridizes under stringentconditions to a sequence as set forth in SEQ ID NO:1, or a subsequencethereof; a sequence as set forth in SEQ ID NO:3, or a subsequencethereof; a sequence as set forth in SEQ ID NO:5, or a subsequencethereof; or, a sequence as set forth in SEQ ID NO:7, or a subsequencethereof.

The invention provides methods for identifying a modulator of aphospholipase activity comprising the following steps: (a) providing apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing a test compound; (c) contacting thepolypeptide of step (a) with the test compound of step (b); and,measuring an activity of the phospholipase, wherein a change in thephospholipase activity measured in the presence of the test compoundcompared to the activity in the absence of the test compound provides adetermination that the test compound modulates the phospholipaseactivity. In alternative aspects, the nucleic acid can have at least 85%sequence identity to SEQ ID NO:1 over a region of at least about 100residues, at least 80% sequence identity to SEQ ID NO:3 over a region ofat least about 100 residues, at least 80% sequence identity to SEQ IDNO:5 over a region of at least about 100 residues, or, at least 70%sequence identity to SEQ ID NO:7 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection. Inalternative aspects, the nucleic acid can hybridize under stringentconditions to a nucleic acid sequence selected from the group consistingof a sequence as set forth in SEQ ID NO:1, or a subsequence thereof; asequence as set forth in SEQ ID NO:3, or a subsequence thereof; asequence as set forth in SEQ ID NO:5, or a subsequence thereof; and, asequence as set forth in SEQ ID NO:7, or a subsequence thereof.

In one aspect, the phospholipase activity is measured by providing aphospholipase substrate and detecting an increase in the amount of thesubstrate or a decrease in the amount of a reaction product. Thedecrease in the amount of the substrate or the increase in the amount ofthe reaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of phospholipase activity. The increase inthe amount of the substrate or the decrease in the amount of thereaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an inhibitor of phospholipase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence of the invention or a nucleic acid sequence ofthe invention.

In one aspect, the computer system can further comprise a sequencecomparison algorithm and a data storage device having at least onereference sequence stored thereon. The sequence comparison algorithm cancomprise a computer program that indicates polymorphisms. The computersystem can further comprising an identifier that identifies one or morefeatures in said sequence.

The invention provides computer readable mediums having stored thereon asequence comprising a polypeptide sequence of the invention or a nucleicacid sequence of the invention.

The invention provides methods for identifying a feature in a sequencecomprising the steps of: (a) reading the sequence using a computerprogram which identifies one or more features in a sequence, wherein thesequence comprises a polypeptide sequence of the invention or a nucleicacid sequence of the invention; and, (b) identifying one or morefeatures in the sequence with the computer program.

The invention provides methods for comparing a first sequence to asecond sequence comprising the steps of: (a) reading the first sequenceand the second sequence through use of a computer program which comparessequences, wherein the first sequence comprises a polypeptide sequenceof the invention or a nucleic acid sequence of the invention; and, (b)determining differences between the first sequence and the secondsequence with the computer program. In one aspect, the step ofdetermining differences between the first sequence and the secondsequence further comprises the step of identifying polymorphisms. In oneaspect, the method further comprises an identifier (and use of theidentifier) that identifies one or more features in a sequence. In oneaspect, the method comprises reading the first sequence using a computerprogram and identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide with a phospholipase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide with a phospholipase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention (e.g., SEQID NO:1, or a subsequence thereof; SEQ ID NO:3, or a subsequencethereof; SEQ ID NO:5, or a subsequence thereof; or SEQ ID NO:7, or asubsequence thereof, etc.); (b) isolating a nucleic acid from theenvironmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to theamplification primer pair; and, (c) combining the nucleic acid of step(b) with the amplification primer pair of step (a) and amplifyingnucleic acid from the environmental sample, thereby isolating orrecovering a nucleic acid encoding a polypeptide with a phospholipaseactivity from an environmental sample. In one aspect, each member of theamplification primer sequence pair comprises an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a nucleic acidsequence of the invention. In one aspect, the amplification primersequence pair is an amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide with a phospholipase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid sequence of theinvention, or a subsequence thereof; (b) isolating a nucleic acid fromthe environmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to apolynucleotide probe of step (a); (c) combining the isolated nucleicacid or the treated environmental sample of step (b) with thepolynucleotide probe of step (a); and, (d) isolating a nucleic acid thatspecifically hybridizes with the polynucleotide probe of step (a),thereby isolating or recovering a nucleic acid encoding a polypeptidewith a phospholipase activity from the environmental sample. Inalternative aspects, the environmental sample comprises a water sample,a liquid sample, a soil sample, an air sample or a biological sample. Inalternative aspects, the biological sample is derived from a bacterialcell, a protozoan cell, an insect cell, a yeast cell, a plant cell, afungal cell, an algal (algae) cell, a lichen, or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a phospholipase comprising the steps of: (a) providing atemplate nucleic acid comprising a nucleic acid of the invention; (b)modifying, deleting or adding one or more nucleotides in the templatesequence, or a combination thereof, to generate a variant of thetemplate nucleic acid.

In one aspect, the method further comprises expressing the variantnucleic acid to generate a variant phospholipase polypeptide. Inalternative aspects, the modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis™ (GSSM™), synthetic ligation reassembly(SLR) and/or a combination thereof. In alternative aspects, themodifications, additions or deletions are introduced by a methodselected from the group consisting of recombination, recursive sequencerecombination, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesis,chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and/or a combination thereof.

In one aspect, the method is iteratively repeated until a phospholipasehaving an altered or different activity or an altered or differentstability from that of a phospholipase encoded by the template nucleicacid is produced. In one aspect, the altered or different activity is aphospholipase activity under an acidic condition, wherein thephospholipase encoded by the template nucleic acid is not active underthe acidic condition. In one aspect, the altered or different activityis a phospholipase activity under a high temperature, wherein thephospholipase encoded by the template nucleic acid is not active underthe high temperature. In one aspect, the method is iteratively repeateduntil a phospholipase coding sequence having an altered codon usage fromthat of the template nucleic acid is produced. The method can beiteratively repeated until a phospholipase gene having higher or lowerlevel of message expression or stability from that of the templatenucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a phospholipase to increase its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodinga phospholipase; and, (b) identifying a non-preferred or a lesspreferred codon in the nucleic acid of step (a) and replacing it with apreferred or neutrally used codon encoding the same amino acid as thereplaced codon, wherein a preferred codon is a codon over-represented incoding sequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a phospholipase, the method comprising (a) providing a nucleicacid of the invention encoding a phospholipase; and, (b) identifying acodon in the nucleic acid of step (a) and replacing it with a differentcodon encoding the same amino acid as the replaced codon, therebymodifying codons in a nucleic acid encoding a phospholipase.

The invention provides methods for modifying codons in a nucleic acidencoding a phospholipase to increase its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodinga phospholipase; and, (b) identifying a non-preferred or a lesspreferred codon in the nucleic acid of step (a) and replacing it with apreferred or neutrally used codon encoding the same amino acid as thereplaced codon, wherein a preferred codon is a codon over-represented incoding sequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a phospholipase to decrease its expression in a host cell, themethod comprising (a) providing a nucleic acid of the invention encodinga phospholipase; and, (b) identifying at least one preferred codon inthe nucleic acid of step (a) and replacing it with a non-preferred orless preferred codon encoding the same amino acid as the replaced codon,wherein a preferred codon is a codon over-represented in codingsequences in genes in a host cell and a non-preferred or less preferredcodon is a codon under-represented in coding sequences in genes in thehost cell, thereby modifying the nucleic acid to decrease its expressionin a host cell. In alternative aspects, the host cell is a bacterialcell, a fungal cell, an insect cell, a yeast cell, a plant cell, analgal (algae) cell, a lichen, or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified phospholipase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising: (a) providing a first nucleic acid encoding a firstactive site or first substrate binding site, wherein the first nucleicacid sequence comprises a nucleic acid of the invention; (b) providing aset of mutagenic oligonucleotides that encode naturally-occurring aminoacid variants at a plurality of targeted codons in the first nucleicacid; and, (c) using the set of mutagenic oligonucleotides to generate aset of active site-encoding or substrate binding site-encoding variantnucleic acids encoding a range of amino acid variations at each aminoacid codon that was mutagenized, thereby producing a library of nucleicacids encoding a plurality of modified phospholipase active sites orsubstrate binding sites. In alternative aspects, the method comprisesmutagenizing the first nucleic acid of step (a) by a method comprisingan optimized directed evolution system, Gene Site SaturationMutagenesis™ (GSSM™), and synthetic ligation reassembly (SLR). Themethod can further comprise mutagenizing the first nucleic acid of step(a) or variants by a method comprising error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, Gene Site Saturation Mutagenesis™ (GSSM™),synthetic ligation reassembly (SLR) and a combination thereof. Themethod can further comprise mutagenizing the first nucleic acid of step(a) or variants by a method comprising recombination, recursive sequencerecombination, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesis,chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe steps of: (a) providing a plurality of biosynthetic enzymes capableof synthesizing or modifying a small molecule, wherein one of theenzymes comprises a phospholipase enzyme encoded by a nucleic acid ofthe invention; (b) providing a substrate for at least one of the enzymesof step (a); and, (c) reacting the substrate of step (b) with theenzymes under conditions that facilitate a plurality of biocatalyticreactions to generate a small molecule by a series of biocatalyticreactions.

The invention provides methods for modifying a small molecule comprisingthe steps: (a) providing a phospholipase enzyme encoded by a nucleicacid of the invention; (b) providing a small molecule; and, (c) reactingthe enzyme of step (a) with the small molecule of step (b) underconditions that facilitate an enzymatic reaction catalyzed by thephospholipase enzyme, thereby modifying a small molecule by aphospholipase enzymatic reaction. In one aspect, the method comprisesproviding a plurality of small molecule substrates for the enzyme ofstep (a), thereby generating a library of modified small moleculesproduced by at least one enzymatic reaction catalyzed by thephospholipase enzyme. In one aspect, the method further comprises aplurality of additional enzymes under conditions that facilitate aplurality of biocatalytic reactions by the enzymes to form a library ofmodified small molecules produced by the plurality of enzymaticreactions. In one aspect, the method further comprises the step oftesting the library to determine if a particular modified small moleculethat exhibits a desired activity is present within the library. The stepof testing the library can further comprises the steps of systematicallyeliminating all but one of the biocatalytic reactions used to produce aportion of the plurality of the modified small molecules within thelibrary by testing the portion of the modified small molecule for thepresence or absence of the particular modified small molecule with adesired activity, and identifying at least one specific biocatalyticreaction that produces the particular modified small molecule of desiredactivity.

The invention provides methods for determining a functional fragment ofa phospholipase enzyme comprising the steps of: (a) providing aphospholipase enzyme comprising an amino acid sequence of the invention;and, (b) deleting a plurality of amino acid residues from the sequenceof step (a) and testing the remaining subsequence for a phospholipaseactivity, thereby determining a functional fragment of a phospholipaseenzyme. In one aspect, the phospholipase activity is measured byproviding a phospholipase substrate and detecting an increase in theamount of the substrate or a decrease in the amount of a reactionproduct. In one aspect, a decrease in the amount of an enzyme substrateor an increase in the amount of the reaction product with the testcompound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an activatorof phospholipase activity.

The invention provides methods for cleaving a glycerolphosphate esterlinkage comprising the following steps: (a) providing a polypeptidehaving a phospholipase activity, wherein the polypeptide comprises anamino acid sequence of the invention, or the polypeptide is encoded by anucleic acid of the invention; (b) providing a composition comprising aglycerolphosphate ester linkage; and, (c) contacting the polypeptide ofstep (a) with the composition of step (b) under conditions wherein thepolypeptide cleaves the glycerolphosphate ester linkage. In one aspect,the conditions comprise between about pH 5 to about 8.5, or, betweenabout pH 4.5 (or more acidic, i.e., pH<4.5) to about 9.0 (or morealkaline (i.e., pH>9). In one aspect, the conditions comprise atemperature of between about 40° C. and about 70° C. In one aspect, thecomposition comprises a vegetable oil. In one aspect, the compositioncomprises an oilseed phospholipid. In one aspect, the cleavage reactioncan generate a water extractable phosphorylated base and a diglyceride.

The invention provides methods hydrolyzing, breaking up or disrupting aphospholipid-comprising composition comprising providing at least onepolypeptide of the invention having a phospholipase activity, or apolypeptide having a phospholipase activity encoded by at least onenucleic acid of the invention; providing a composition comprising aphospholipid; and contacting the polypeptide with the composition underconditions wherein the phospholipase hydrolyzes, breaks up or disruptsthe phospholipid-comprising composition. In one aspect, the methodcomprises use of high shear mixing of the composition, followed by no orlow shear mixing with the at least one polypeptide of the inventionhaving a phospholipase activity to allow adequate “contacting” of thephospholipid substrate with the phospholipase. The at least onepolypeptide having a phospholipase activity can also be present in thehigh shear mixing step. The process can be practiced at any scale, e.g.,at a scale comprising about 1 gram (g) to about 500, 1000, 2000, 2500,5000 g, or more, or any amount in this range.

The invention provides methods for oil degumming comprising thefollowing steps: (a) providing at least one polypeptide having aphospholipase activity, wherein the polypeptide comprises an amino acidsequence of the invention, or the polypeptide is encoded by a nucleicacid of the invention; (b) providing a composition comprising avegetable oil; and, (c) contacting the polypeptide of step (a) and thevegetable oil of step (b) under conditions wherein the polypeptide cancleave ester linkages in the vegetable oil, thereby degumming the oil.In one aspect, the vegetable oil comprises oilseed. The vegetable oilcan comprise rice bran oils, palm oil, rapeseed oil, corn oil, soybeanoil, canola oil, sesame oil, peanut oil or sunflower oil. In one aspect,the method further comprises addition of a phospholipase of theinvention, another phospholipase or a combination thereof. In oneaspect, more than one polypeptide having a phospholipase activity isadded to the process, wherein at least one polypeptide is an enzyme ofthe invention. In one aspect, the enzymes are added in a specific order,e.g., PLCs with differing specificities in are added in a specificorder, for example, an enzyme with PC and PE activity is added first (ortwo enzymes are added together, one with PC and the other with PEactivity), then an enzyme with PI PLC activity is added, or anycombination thereof.

In one aspect of the oil degumming process, the oil-comprisingcomposition comprises a plant, an animal, an algae or a fish oil or fat.The plant oil can comprise a rice bran oil, a soybean oil, a rapeseedoil, a corn oil, an oil from a palm kernel, a canola oil, a sunfloweroil, a sesame oil or a peanut oil. The polypeptide can hydrolyze aphosphatide from a hydratable and/or a non-hydratable phospholipid inthe oil-comprising composition. In one aspect, the polypeptidehydrolyzes a phosphatide at a glyceryl phosphoester bond to generate adiglyceride and water-soluble phosphate compound. In one aspect, thepolypeptide has a phospholipase C activity. In one aspect, thepolypeptide is a phospholipase D and a phosphatase enzyme is also added.

In one aspect of the oil degumming process, the contacting compriseshydrolysis of a hydrated phospholipid in an oil. The hydrolysisconditions can comprise alkaline conditions, e.g., in one aspect, theconditions comprise a temperature of about 20° C. to 40° C. at thealkaline pH. The alkaline conditions can comprise a pH of about pH 8 topH 10, or more. The hydrolysis conditions can be made alkaline at anytime in the process, e.g., in one aspect, a phospholipase, such as aPLC, is added before the conditions are made alkaline (e.g., a “causticneutralization” of an acid-comprising oil, such as phosphatidic acid).

In one aspect of the oil degumming process, the base causes theisomerization of 1,2-DAG, produced by PLC, into 1,3-DAG which provides anutritional health benefit over 1,2-DAG, e.g., the 1,3-DAG is burned asenergy instead of being stored as fat (as is 1,2-DAG). Thus, theinvention provides a caustic oil refining process wherein aphospholipase, e.g., an enzyme of the invention, including a PLC, isadded “at the front end”, i.e., before adding any acid and caustic,e.g., as illustrated in the exemplary process of FIG. 13. One of theconsequences of adding the PLC at the front end of a caustic refiningprocess of the invention (see further discussion, below), and adding theacid and caustic subsequently, is the generation of an elevated level of1,3-DAG (not 1,2-DAG). This may be a consequence of acid orbase-catalyzed acyl migration. Nutritionally, 1,3-DAG is better than1,2-DAG. Thus, the invention comprises an oil degumming process using aPLC of the invention, whereby the final degummed oil product containsnot less than about 0.5%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0% 1,3-DAG.

In one aspect of the oil degumming process, the hydrolysis conditionscan comprise a reaction time of about 3 to 10 or more minutes. Thehydrolysis conditions can comprise hydrolysis of hydratable andnon-hydratable phospholipids in oil at a temperature of between about50° C. to 60° C., at a pH of between about pH 5 to pH 6.5, or betweenabout pH 5 to pH 7.5, or between about pH 5 to pH 8.0, using a reactiontime of about 30 to 60 minutes.

In one aspect of the oil degumming process, the polypeptide is bound toa filter and the phospholipid-containing fat or oil is passed throughthe filter. The polypeptide can be added to a solution comprising thephospholipid-containing fat or oil and then the solution is passedthrough a filter.

In one aspect the oil degumming method further comprises physicalremoval of gum produced by the degumming process by addition of ahardening substance, e.g., a talc or equivalent. In one aspect, thisincreases oil gain.

The invention also provides methods for converting a non-hydratablephospholipid to a hydratable form comprising the following steps: (a)providing a polypeptide having a phospholipase activity, wherein thepolypeptide comprises an amino acid sequence of the invention, or thepolypeptide is encoded by a nucleic acid of the invention; (b) providinga composition comprising a non-hydratable phospholipid; and, (c)contacting the polypeptide of step (a) and the non-hydratablephospholipid of step (b) under conditions wherein the polypeptide cancleave ester linkages in the non-hydratable phospholipid, therebyconverting a non-hydratable phospholipid to a hydratable form.

The invention provides methods for degumming an oil comprising thefollowing steps: (a) providing a composition comprising a polypeptide ofthe invention having a phospholipase activity or a polypeptide encodedby a nucleic acid of the invention; (b) providing an compositioncomprising a fat or an oil comprising a phospholipid; and (c) contactingthe polypeptide of step (a) and the composition of step (b) underconditions wherein the polypeptide can degum the phospholipid-comprisingcomposition (under conditions wherein the polypeptide of the inventioncan catalyze the hydrolysis of a phospholipid). In one aspect theoil-comprising composition comprises a plant, an animal, an algae or afish oil. The plant oil can comprise a rice bran oil, a soybean oil, arapeseed oil, a corn oil, an oil from a palm kernel, a canola oil, asunflower oil, a sesame oil or a peanut oil. The polypeptide canhydrolyze a phosphatide from a hydratable and/or a non-hydratablephospholipid in the oil-comprising composition. The polypeptide canhydrolyze a phosphatide at a glyceryl phosphoester bond to generate adiglyceride and water-soluble phosphate compound. The polypeptide canhave a phospholipase C, B, A or D activity. In one aspect, aphospholipase D activity and a phosphatase enzyme are added. Thecontacting can comprise hydrolysis of a hydrated phospholipid in an oil.The hydrolysis conditions of can comprise a temperature of about 20° C.to 40° C. at an alkaline pH. The alkaline conditions can comprise a pHof about pH 8 to pH 10. The hydrolysis conditions can comprise areaction time of about 3 to 10 minutes. The hydrolysis conditions cancomprise hydrolysis of hydratable and non-hydratable phospholipids inoil at a temperature of about 50° C. to 60° C., at a pH of about pH 5 topH 6.5 using a reaction time of about 30 to 60 minutes. The polypeptidecan be bound to a filter and the phospholipid-containing fat or oil ispassed through the filter. The polypeptide can be added to a solutioncomprising the phospholipid-containing fat or oil and then the solutionis passed through a filter.

The invention provides methods for converting a non-hydratablephospholipid to a hydratable form comprising the following steps: (a)providing a composition comprising a polypeptide having a phospholipaseactivity of the invention, or a polypeptide encoded by a nucleic acid ofthe invention; (b) providing an composition comprising a non-hydratablephospholipid; and (c) contacting the polypeptide of step (a) and thecomposition of step (b) under conditions wherein the polypeptideconverts the non-hydratable phospholipid to a hydratable form. Thepolypeptide can have a phospholipase C activity. The polypeptide canhave a phospholipase D activity and a phosphatase enzyme is also added.

The invention provides methods for caustic refining of aphospholipid-containing composition comprising the following steps: (a)providing a composition comprising a phospholipase, which can be apolypeptide of the invention having a phospholipase activity, or apolypeptide encoded by a nucleic acid of the invention; (b) providing ancomposition comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) before, duringor after the caustic refining. The polypeptide can have a phospholipaseactivity, e.g., PLC, PLB, PLD and/or PLA activity. The polypeptide canbe added before caustic refining, i.e., at the “front end” of theprocess, before adding acid or caustic, as illustrated in FIG. 13.

The polypeptide (which can be an enzyme, e.g., a PLC, of the invention)can be added during caustic refining and varying levels of acid andcaustic can be added depending on levels of phosphorus and levels offree fatty acids. The polypeptide (which can be an enzyme of theinvention) can be added before caustic refining, or, after causticrefining: in an intense mixer or retention mixer prior to separation;following a heating step; in a centrifuge; in a soapstock; in awashwater; and/or, during bleaching or deodorizing steps. The method cancomprise use of concentrated solutions of caustic, e.g., moreconcentrated than the industrial standard of 11%, to decrease mass ofgum. In alternative aspects, the concentrated solution of caustic isbetween about 12% and 50% concentrated, e.g., about 20%, 30%, 40%, 50%or 60%, or more, concentrated.

The composition comprising the phospholipid can comprise a plant. Thepolypeptide can be expressed transgenically in the plant. Thepolypeptide having a phospholipase activity can be added during crushingof a seed or other plant part, or, the polypeptide having aphospholipase activity is added following crushing or prior to refining.

Also provided is a caustic refining process for hydrolyzingphospholipids in oil (e.g., plant oil) using a polypeptide of theinvention to generate diacylglycerol (DAG) and water-soluble phosphateester. In one aspect, the enzyme of the invention must operate in acaustic refining process, including, optionally low water and/or in atemperature range of about 55° C. to about 70° C. Use of a causticrefining process with low water in this temperature range will maximizeyield by increasing DAG and reducing entrained oil. In one aspect, theenzyme used in this caustic refining process of the invention has bothvery good activity on phosphatidylcholine (PC) andphosphatidylethanolamine (PE), is active between a pH of about pH 6 topH 9, is active up to 75° C., and is active in low water in oil, e.g.,about 2% to 5% water, e.g., the enzyme encoded by the sequence of SEQ IDNO:2, encoded e.g., by SEQ ID NO: 1.

In another aspect of the invention's caustic refining process forhydrolyzing phospholipids in oils, two enzymes are used: a PI-specificPLC (hydrolyzes PI), and a PC-PLC that hydrolyzes PC, PE and PA. Thisembodiment generates oil suitable for chemical or physical refining andmaximizes yield increase from DAG and less entrained oil.

The invention provides methods for purification of a phytosterol or atriterpene comprising the following steps: (a) providing a compositioncomprising a polypeptide of the invention having a phospholipaseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing an composition comprising a phytosterol or a triterpene;and (c) contacting the polypeptide of step (a) with the composition ofstep (b) under conditions wherein the polypeptide can catalyze thehydrolysis of a phospholipid in the composition. The polypeptide canhave a phospholipase C activity. The phytosterol or a triterpene cancomprise a plant sterol. The plant sterol can be derived from avegetable oil. The vegetable oil can comprise a rice bran oil, a coconutoil, canola oil, cocoa butter oil, corn oil, cottonseed oil, linseedoil, olive oil, palm oil, peanut oil, oil derived from a rice bran,safflower oil, sesame oil, soybean oil or a sunflower oil. The methodcan comprise use of nonpolar solvents to quantitatively extract freephytosterols and phytosteryl fatty-acid esters. The phytosterol or atriterpene can comprise a β-sitosterol, a campesterol, a stigmasterol, astigmastanol, a β-sitostanol, a sitostanol, a desmosterol, achalinasterol, a poriferasterol, a clionasterol or a brassicasterol.

The invention provides methods for refining a crude oil comprising thefollowing steps: (a) providing a composition comprising a polypeptide ofthe invention having a phospholipase activity, or a polypeptide encodedby a nucleic acid of the invention; (b) providing a compositioncomprising an oil comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide can catalyze the hydrolysis of aphospholipid in the composition. The polypeptide can have aphospholipase C activity. The polypeptide can have a phospholipaseactivity is in a water solution that is added to the composition. Thewater level can be between about 0.5 to 5%. The process time can be lessthan about 2 hours, less than about 60 minutes, less than about 30minutes, less than 15 minutes, or less than 5 minutes. The hydrolysisconditions can comprise a temperature of between about 25° C.-70° C. Thehydrolysis conditions can comprise use of caustics. Concentratedsolutions of caustic, e.g., more concentrated than the industrialstandard of 11%, to decrease mass of gum can be used. In alternativeaspects, the concentrated solution of caustic is between about 12% and50% concentrated, e.g., about 20%, 30%, 40%, 50%, or 60% or moreconcentrated.

The hydrolysis conditions can comprise a pH of between about pH 3 and pH10, between about pH 4 and pH 9, or between about pH 5 and pH 8. Thehydrolysis conditions can comprise addition of emulsifiers and/or mixingafter the contacting of step (c). The methods can comprise addition ofan emulsion-breaker and/or heat or cooling (e.g. to between about 4° C.to about −20° C., or less) to promote separation of an aqueous phase.The methods can comprise degumming before the contacting step to collectlecithin by centrifugation and then adding a PLC, a PLC and/or a PLA toremove non-hydratable phospholipids. The methods can comprise waterdegumming of crude oil to less than 10 ppm phosphorus for edible oilsand subsequent physical refining to less than about 50 ppm phosphorusfor biodiesel oils. The methods can comprise addition of acid to promotehydration of non-hydratable phospholipids. In one aspect, addition ofacid promotes lowering of the calcium and magnesium metal content.

The invention provides a method for ameliorating or preventinglipopolysaccharide (LPS)-mediated toxicity comprising administering to apatient a pharmaceutical composition comprising a polypeptide of theinvention. The invention provides a method for detoxifying an endotoxincomprising contacting the endotoxin with a polypeptide of the invention.The invention provides a method for deacylating a 2′ or a 3′ fatty acidchain from a lipid A comprising contacting the lipid A with apolypeptide of the invention.

The invention provides a method for refining a lubricant comprising thefollowing steps: (a) providing a composition comprising an enzyme of theinvention; (b) providing a lubricant; and (c) treating the lubricantwith an enzyme under conditions wherein the enzyme can selectivehydrolyze oils in the lubricant, thereby refining it. The lubricant canbe a hydraulic oil.

The invention provides a method of treating a fabric comprising thefollowing steps: (a) providing a composition comprising an enzyme of theinvention, (b) providing a fabric; and (c) treating the fabric with theenzyme. The treatment of the fabric can comprise improvement of the handand drape of the final fabric, dyeing, obtaining flame retardancy,obtaining water repellency, obtaining optical brightness, or obtainingresin finishing. The fabric can comprise cotton, viscose, rayon,lyocell, flax, linen, ramie, all blends thereof, or blends thereof withpolyesters, wool, polyamides acrylics or polyacrylics. The inventionprovides a fabric, yarn or fiber comprising an enzyme of the invention.The enzyme can be adsorbed, absorbed or immobilized on the surface ofthe fabric, yarn or fiber.

The invention provides methods for expressing phospholipase C comprisingproviding a Pichia strain with a Mut⁺ phenotype; inserting aheterologous phospholipase C-encoding nucleic acid in the Pichia strain;and, culturing the Pichia strain under conditions whereby thephospholipase C is expressed. The method can further comprisesupplementing the culture conditions with zinc. The invention alsoprovides cell systems, isolated cells and cell lines for expressingphospholipase C comprising a Mut⁺ phenotype Pichia strain comprising aheterologous phospholipase C-encoding nucleic acid operably linked to apromoter operable in the Pichia strain.

The invention provides zeocin-resistant yeast cell systems (e.g., yeastcells, cell lines, individual cells) for expressing a heterologousprotein comprising the steps of providing a Pichia sp. (e.g., P.pastoris) cell comprising a heterologous nucleic acid capable ofexpressing a heterologous protein; culturing the cell under conditionscomprising zeocin at an initial concentration; selecting cells resistantto the initial concentration of zeocin, and reculturing under conditionscomprising a higher concentration of zeocin; and selecting the cellscultured in step (c) resistant to the higher concentration of zeocin. Inone aspect, the heterologous protein is an enzyme, or optionally, aphospholipase, or optionally a phospholipase C (PLC), e.g., any enzymeof the invention.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is a block diagram of a computer system, as described in detail,below.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database, as described in detail,below.

FIG. 3 is a flow diagram illustrating one embodiment of a process in acomputer for determining whether two sequences are homologous, asdescribed in detail, below.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess for detecting the presence of a feature in a sequence, asdescribed in detail, below.

FIGS. 5A, 5B and 5C schematically illustrate a model two-phase systemfor simulation of PLC-mediated degumming, as described in detail inExample 2, below.

FIG. 6 schematically illustrates an exemplary vegetable oil refiningprocess using the phospholipases of the invention.

FIG. 7 schematically illustrates an exemplary degumming process of theinvention for physically refined oils, as discussed in detail, below.

FIG. 8 schematically illustrates phosphatide hydrolysis with aphospholipase C of the invention, as discussed in detail, below.

FIG. 9 schematically illustrates an exemplary caustic refining processof the invention, and illustrates an alternative embodiment comprisingapplication of a phospholipase C of the invention as a “Caustic RefiningAid” (Long Mix Caustic Refining), as discussed in detail, below.

FIG. 10 schematically illustrates application of a phospholipase C ofthe invention as a degumming aid, as discussed in detail, below.

FIG. 11 is a chart describing selected characteristics of exemplarynucleic acids and polypeptides of the invention, as described in furtherdetail, below.

FIG. 12 schematically illustrates data from a two enzyme system of theinvention, as described in Example 3, below.

FIG. 13 schematically illustrates an exemplary caustic refining processof the invention, and illustrates an alternative embodiment comprisingapplication of a phospholipase C of the invention as a “Caustic RefiningAid” (Long Mix Caustic Refining), as discussed in detail, below.

FIG. 14 illustrates another variation of methods of the invention wheretwo centrifugation steps are used in the process, as discussed indetail, below.

FIG. 15 illustrates another variation of methods of the invention wherethree centrifugation steps are used in the process, as discussed indetail, below.

FIG. 16 illustrates another exemplary variation of this process usingacid treatment and having a centrifugation step before a degumming step,as discussed in detail, below.

FIG. 17 illustrates the results of the in vitro digestion experimentswherein the phospholipase C variants of the invention, as discussed indetail in Example 4, below.

FIG. 18 illustrates the results of a batch fermentor culture using anexemplary enzyme of the invention, as discussed in detail in Example 5,below.

FIG. 19 illustrates the results of Oxygen Uptake Rate (“OUR”)comparisons of cultures of P. pastoris MutS strains of the invention, asdiscussed in detail in Example 5, below.

FIG. 20 illustrates a methanol consumption profile comparison in P.pastoris MutS strains of the invention, as discussed in detail inExample 5, below.

FIG. 21 illustrates an “OUR” profile of a culture of a recombinant formof the exemplary PLC enzyme of the invention SEQ ID NO:2, as discussedin detail in Example 5, below.

FIG. 22 illustrates results from an SDS-PAGE showing the quality of PLCprotein produced in a culture, and a corresponding OUR profile, of aculture of a recombinant form of the exemplary PLC enzyme of theinvention SEQ ID NO:2, as discussed in detail in Example 5, below.

FIG. 23 illustrates results from an SDS-PAGE showing the quantity ofactive PLC located intracellularly in a culture of a recombinant form ofthe exemplary PLC enzyme of the invention SEQ ID NO:2, as discussed indetail in Example 5, below.

FIG. 24 illustrates a visualization of the morphological changes inyeast cells associated with active PLC—a recombinant form of theexemplary PLC enzyme of the invention SEQ ID NO:2, as discussed indetail in Example 5, below.

FIG. 25 graphically summarizes data showing the status of a PLCproduction performance at 95 h TFT (total fermentation time) in Pichiausing an exemplary PLC enzyme of the invention SEQ ID NO:2, as discussedin detail in Example 5, below.

FIG. 26 is a table summary of data from expression screening ofexemplary zeocin-adapted cell colonies of the invention, as discussed indetail in Example 5, below.

FIG. 27 illustrates data showing that PLC protein levels were higher incultures comprising exemplary zeocin-adapted cell colonies of theinvention, as discussed in detail in Example 5, below.

FIG. 28 illustrates data showing a growth comparison of zeo-adaptedcolonies of the invention vs control, as discussed in detail in Example5, below.

FIG. 29 illustrates the results of a heating experiment demonstratingthe thermostability of the exemplary enzyme of the invention SEQ IDNO:2, with the conditions indicated in the figure, as discussed indetail in Example 6, below.

FIG. 30 illustrates NMR data summarizing the heating experimentdemonstrating the thermostability of the exemplary enzyme of theinvention SEQ ID NO:2, as discussed in detail in Example 6, below.

FIGS. 31, 32 and 33 illustrate data demonstrating the thermal stabilityof SEQ ID NO:2 using p-NPPC, at the conditions shown in the figure, asdiscussed in detail in Example 6, below.

FIG. 34 illustrates data demonstrating the thermal stability of SEQ IDNO:2 using DSC analysis, as discussed in detail in Example 6, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides phospholipases, e.g., polypeptides havingphospholipase A, B, C, D, patatin, phosphatidic acid phosphatases (PAP)and/or lipid acyl hydrolase (LAH) or equivalent activity,polynucleotides encoding them and methods for making and using them. Theinvention provides enzymes that efficiently cleave glycerolphosphateester linkage in oils, such as vegetable oils, e.g., oilseedphospholipids, to generate a water extractable phosphorylated base and adiglyceride. In one aspect, the phospholipases of the invention have alipid acyl hydrolase (LAH) activity. In alternative aspects, thephospholipases of the invention can cleave glycerolphosphate esterlinkages in phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid,and/or sphingomyelin, or a combination thereof. For example, in oneaspect a phospholipase of the invention is specific for one or morespecific substrates, e.g., an enzyme of the invention can have aspecificity of action for PE and PC; PE an PI; PE and PS; PS and PE; PSand PI; PI and PE; PS, PI and PC; PE, PI and PC; or, PE, PS, PI and PC.

A phospholipase of the invention (e.g., polypeptides havingphospholipase A, B, C, D, patatin, phosphatidic acid phosphatases (PAP)and/or lipid acyl hydrolase (LAH) or equivalent activity) can be usedfor enzymatic degumming of vegetable oils because the phosphate moietyis soluble in water and easy to remove. The diglyceride product willremain in the oil and therefore will reduce losses. The PLCs of theinvention can be used in addition to or in place of PLA1s and PLA2s incommercial oil degumming, such as in the ENZYMAX® process, wherephospholipids are hydrolyzed by PLA1 and PLA2.

In one aspect, the phospholipases of the invention are active at a highand/or at a low temperature, or, over a wide range of temperature, e.g.,they can be active in the temperatures ranging between 20° C. to 90° C.,between 30° C. to 80° C., or between 40° C. to 70° C. The invention alsoprovides phospholipases of the invention have activity at alkaline pHsor at acidic pHs, e.g., low water acidity. In alternative aspects, thephospholipases of the invention can have activity in acidic pHs as lowas pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0 and pH 3.5 or moreacidic (i.e., <pH 3.5). In alternative aspects, the phospholipases ofthe invention can have activity in alkaline pHs as high as pH 7.5, pH8.0, pH 8.5, pH 9.0, pH 9.5, pH 10 or more alkaline (i.e., >pH 10). Inone aspect, the phospholipases of the invention are active in thetemperature range of between about 40° C. to about 70° C., 75° C., or80° C., or more, under conditions of low water activity (low watercontent).

The invention also provides methods for further modifying the exemplaryphospholipases of the invention to generate enzymes with desirableproperties. For example, phospholipases generated by the methods of theinvention can have altered substrate specificities, substrate bindingspecificities, substrate cleavage patterns, thermal stability,pH/activity profile, pH/stability profile (such as increased stabilityat low, e.g. pH<6 or pH<5, or high, e.g. pH>9, pH values), stabilitytowards oxidation, Ca²⁺ dependency, specific activity and the like. Theinvention provides for altering any property of interest. For instance,the alteration may result in a variant which, as compared to a parentphospholipase, has altered pH and temperature activity profile.

In one aspect, the phospholipases of the invention are used in variousvegetable oil processing steps, such as in vegetable oil extraction,particularly, in the removal of “phospholipid gums” in a process called“oil degumming,” as described herein. The invention providescompositions (e.g., comprising enzymes of the invention) and processesfor the production of vegetable oils from various sources, such as oilfrom rice bran, soybeans, rapeseed, peanut, sesame, sunflower and corn.The phospholipase enzymes of the invention can be used in place of PLA,e.g., phospholipase A2, in any vegetable oil processing step.

DEFINITIONS

The term “phospholipase” encompasses enzymes having any phospholipaseactivity, for example, cleaving a glycerolphosphate ester linkage(catalyzing hydrolysis of a glycerolphosphate ester linkage), e.g., inan oil, such as a vegetable oil. The phospholipase activity of theinvention can generate a water extractable phosphorylated base and adiglyceride. The phospholipase activity of the invention also includeshydrolysis of glycerolphosphate ester linkages at high temperatures, lowtemperatures, alkaline pHs and at acidic pHs. The term “a phospholipaseactivity” also includes cleaving a glycerolphosphate ester to generate awater extractable phosphorylated base and a diglyceride. The term “aphospholipase activity” also includes cutting ester bonds of glycerinand phosphoric acid in phospholipids. The term “a phospholipaseactivity” also includes other activities, such as the ability to bind toand hydrolyze a substrate, such as an oil, e.g. a vegetable oil,substrate also including plant and animal phosphatidylcholines,phosphatidyl-ethanolamines, phosphatidylserines and sphingomyelins. Thephospholipase activity can comprise a phospholipase C (PLC) activity; aphospholipase A (PLA) activity, such as a phospholipase A1 orphospholipase A2 activity; a phospholipase B (PLB) activity, such as aphospholipase B1 or phospholipase B2 activity, includinglysophospholipase (LPL) activity and/or lysophospholipase-transacylase(LPTA) activity; a phospholipase D (PLD) activity, such as aphospholipase D1 or a phospholipase D2 activity; and/or a patatinactivity or any combination thereof. The phospholipase activity cancomprise hydrolysis of a glycoprotein, e.g., as a glycoprotein found ina potato tuber or any plant of the genus Solanum, e.g., Solanumtuberosum. The phospholipase activity can comprise a patatin enzymaticactivity, such as a patatin esterase activity (see, e.g., Jimenez (2002)Biotechnol. Prog. 18:635-640). The phospholipase activity can comprise alipid acyl hydrolase (LAH) activity. The phospholipase activity cancomprise being specific for one or more specific substrates, e.g., anenzyme of the invention can have a specificity of action for PE and PC;PE an PI; PE and PS; PS and PE; PS and PI; PI and PE; PS, PI and PC; PE,PI and PC; or, PE, PS, PI and PC, or any combination thereof.

In one aspect, a phospholipase of the invention can have multifunctionalactivity, e.g., a combination of one or more of the enzyme activitiesdescribed herein. For example, in one aspect, a polypeptide of theinvention is enzymatically active, but lacks a lipase activity or lacksany enzymatic activity that affects a neutral oil (triglyceride)fraction. It may be desirable to use such a polypeptide in a particularprocess, e.g., in a degumming process where it is important that theneutral oil fraction not be harmed (diminished, degraded, e.g.,hydrolyzed). Thus, in one aspect, the invention provides a degummingprocess comprising use of a polypeptide of the invention having aphospholipase activity, but not a lipase activity.

In one aspect, PLC phospholipases of the invention utilize (e.g.,catalyze hydrolysis of) a variety of phospholipid substrates includingphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), and/or phosphatidicacid or a combination thereof. In addition, these enzymes can havevarying degrees of activity on the lysophospholipid forms of thesephospholipids. In various aspects, PLC enzymes of the invention may showa preference for phosphatidylcholine and phosphatidylethanolamine assubstrates.

In one aspect, phosphatidylinositol PLC phospholipases of the inventionutilize a variety of phospholipid substrates includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, and phosphatidic acid, or a combination thereof.In addition, these enzymes can have varying degrees of activity on thelysophospholipid forms of these phospholipids. In various aspects,phosphatidylinositol PLC enzymes of the invention may show a preferencefor phosphatidylinositol as a substrate.

In one aspect, patatin enzymes of the invention utilize a variety ofphospholipid substrates including phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, andphosphatidic acid, or a combination thereof. In addition, these enzymescan have varying degrees of activity on the lysophospholipid forms ofthese phospholipids. In various aspects, patatins of the invention arebased on a conservation of amino acid sequence similarity. In variousaspects, these enzymes display a diverse set of biochemical propertiesand may perform reactions characteristic of PLA1, PLA2, PLC, or PLDenzyme classes.

In one aspect, PLD phospholipases of the invention utilize a variety ofphospholipid substrates including phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, andphosphatidic acid, or a combination thereof. In addition, these enzymescan have varying degrees of activity on the lysophospholipid forms ofthese phospholipids. In one aspect, these enzymes are useful forcarrying out transesterification reactions to produce structuredphospholipids.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below.

A “coding sequence of” or a “sequence encodes” a particular polypeptideor protein, is a nucleic acid sequence which is transcribed andtranslated into a polypeptide or protein when placed under the controlof appropriate regulatory sequences.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a phospholipase of theinvention) in a host compatible with such sequences. Expressioncassettes include at least a promoter operably linked with thepolypeptide coding sequence; and, optionally, with other sequences,e.g., transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, e.g., enhancers.“Operably linked” as used herein refers to linkage of a promoterupstream from a DNA sequence such that the promoter mediatestranscription of the DNA sequence. Thus, expression cassettes alsoinclude plasmids, expression vectors, recombinant viruses, any form ofrecombinant “naked DNA” vector, and the like. A “vector” comprises anucleic acid which can infect, transfect, transiently or permanentlytransduce a cell. It will be recognized that a vector can be a nakednucleic acid, or a nucleic acid complexed with protein or lipid. Thevector optionally comprises viral or bacterial nucleic acids and/orproteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). Vectors include, but are not limited to replicons(e.g., RNA replicons, bacteriophages) to which fragments of DNA may beattached and become replicated. Vectors thus include, but are notlimited to RNA, autonomous self-replicating circular or linear DNA orRNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and includes both the expression and non-expressionplasmids. Where a recombinant microorganism or cell culture is describedas hosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

The term “gene” means the segment of DNA involved in producing apolypeptide chain, including, inter alia, regions preceding andfollowing the coding region, such as leader and trailer, promoters andenhancers, as well as, where applicable, intervening sequences (introns)between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA)of genomic or synthetic origin which may be single-stranded ordouble-stranded and may represent a sense or antisense strand, topeptide nucleic acid (PNA), or to any DNA-like or RNA-like material,natural or synthetic in origin, including, e.g., iRNA,ribonucleoproteins (e.g., double stranded iRNAs, e.g., iRNPs). The termencompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones, see e.g., Mata(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soulup (1997)Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid DrugDev 6:153-156.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules.

The terms “polypeptide” and “protein” as used herein, refer to aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain modified amino acids other thanthe 20 gene-encoded amino acids. The term “polypeptide” also includespeptides and polypeptide fragments, motifs and the like. The term alsoincludes glycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, an isolated material or composition can also be a“purified” composition, i.e., it does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library can be conventionally purified toelectrophoretic homogeneity. In alternative aspects, the inventionprovides nucleic acids which have been purified from genomic DNA or fromother sequences in a library or other environment by at least one, two,three, four, five or more orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. In one aspect, nucleic acids represent 5% or moreof the number of nucleic acid inserts in a population of nucleic acid“backbone molecules.” “Backbone molecules” according to the inventioninclude nucleic acids such as expression vectors, self-replicatingnucleic acids, viruses, integrating nucleic acids, and other vectors ornucleic acids used to maintain or manipulate a nucleic acid insert ofinterest. In one aspect, the enriched nucleic acids represent 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the number of nucleic acidinserts in the population of recombinant backbone molecules.“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; e.g., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” refers to either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have at least 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide or amino acidresidue (sequence) identity, when compared and aligned for maximumcorrespondence, as measured using one any known sequence comparisonalgorithm, as discussed in detail below, or by visual inspection. Inalternative aspects, the invention provides nucleic acid and polypeptidesequences having substantial identity to an exemplary sequence of theinvention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, etc., over a region ofat least about 100 residues, 150 residues, 200 residues, 300 residues,400 residues, or a region ranging from between about 50 residues to thefull length of the nucleic acid or polypeptide. Nucleic acid sequencesof the invention can be substantially identical over the entire lengthof a polypeptide coding region.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from a phospholipase polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for phospholipasebiological activity can be removed. Modified polypeptide sequences ofthe invention can be assayed for phospholipase biological activity byany number of methods, including contacting the modified polypeptidesequence with a phospholipase substrate and determining whether themodified polypeptide decreases the amount of specific substrate in theassay or increases the bioproducts of the enzymatic reaction of afunctional phospholipase with the substrate, as discussed further,below.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature, andare well known in the art. For example, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature, altering the timeof hybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a phospholipase of the invention. Variants can be producedby any number of means included methods such as, for example,error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, GSSM™ and anycombination thereof. Techniques for producing variant phospholipaseshaving activity at a pH or temperature, for example, that is differentfrom a wild-type phospholipase, are included herein.

The term “saturation mutagenesis”, Gene Site Saturation Mutagenesis™(GSSM™) or “GSSM™” includes a method that uses degenerateoligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides isolated and recombinant nucleic acids (e.g., theexemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 or SEQ IDNO:173), including expression cassettes such as expression vectors,encoding the polypeptides and phospholipases of the invention. Theinvention also includes methods for discovering new phospholipasesequences using the nucleic acids of the invention. Also provided aremethods for modifying the nucleic acids of the invention by, e.g.,synthetic ligation reassembly, optimized directed evolution systemand/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lac, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lacI promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thephospholipases of the invention. Expression vectors and cloning vehiclesof the invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as Bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector may comprise a promoter, a ribosome-binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures.In general, the DNA sequence is ligated to the desired position in thevector following digestion of the insert and the vector with appropriaterestriction endonucleases. Alternatively, blunt ends in both the insertand the vector may be ligated. A variety of cloning techniques are knownin the art, e.g., as described in Ausubel and Sambrook. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The vector may be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a phospholipase ofthe invention, a vector of the invention. The host cell may be any ofthe host cells familiar to those skilled in the art, includingprokaryotic cells, eukaryotic cells, such as bacterial cells, fungalcells, yeast cells, mammalian cells, insect cells, or plant cells.Enzymes of the invention can be expressed in any host cell, e.g., anybacterial cell, any yeast cell, e.g., Pichia pastoris, Saccharomycescerevisiae or Schizosaccharomyces pombe. Exemplary bacterial cellsinclude E. coli, Lactococcus lactis, Streptomyces, Bacillus subtilis,Bacillus cereus, Salmonella typhimurium or any species within the generaBacillus, Streptomyces and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

An exemplary phospholipase C enzyme (having a sequence as set forth inSEQ ID NO:2) has been over-expressed in active form in a variety of hostsystems including gram negative bacteria, such as E. coli, gram positivebacteria, such as any Bacillus sp. (e.g., Bacillus subtilis, Bacilliscereus), yeast host cells (including, e.g., Pichia pastoris,Saccharomyces sp., such as S. cerevisiae and S. pombe) and Lactococcuslactis, or mammalian, fingi, plant or insect cells. The active enzyme isexpressed from a variety of constructs in each host system. Thesenucleic acid expression constructs can comprise nucleotides encoding thefull-length open reading frame (composed of the signal sequence, thepro-sequence, and the mature protein coding sequence) or they cancomprise a subset of these genetic elements either alone or incombination with heterologous genetic elements that serve as the signalsequence and/or the pro-sequence for the mature open reading frame. Eachof these systems can serve as a commercial production host for theexpression of PLC for use in the previously described enzymatic oildegumming processes.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides ofthe invention, or modified nucleic acids, can be reproduced by, e.g.,amplification. The invention provides amplification primer sequencepairs for amplifying nucleic acids encoding polypeptides with aphospholipase activity. In one aspect, the primer pairs are capable ofamplifying nucleic acid sequences of the invention, e.g., including theexemplary SEQ ID NO:1, or a subsequence thereof; a sequence as set forthin SEQ ID NO:3, or a subsequence thereof; a sequence as set forth in SEQID NO:5, or a subsequence thereof; and, a sequence as set forth in SEQID NO:7, or a subsequence thereof, etc. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a phospholipaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesphospholipases generated by amplification, e.g., polymerase chainreaction (PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a phospholipase by amplification,e.g., polymerase chain reaction (PCR), using an amplification primerpair of the invention. In one aspect, the amplification primer pairamplifies a nucleic acid from a library, e.g., a gene library, such asan environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified. The skilledartisan can select and design suitable oligonucleotide amplificationprimers. Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides isolated and recombinant nucleic acids comprisingsequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary nucleic acidof the invention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ IDNO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 orSEQ ID NO:173, and nucleic acids encoding SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,or SEQ ID NO:174) over a region of at least about 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550 or more, residues. The invention provides polypeptides comprisingsequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary polypeptideof the invention. The extent of sequence identity (homology) may bedetermined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters. In alternative embodiments, thesequence identify can be over a region of at least about 5, 10, 20, 30,40, 50, 100, 150, 200, 250, 300, 350, 400 consecutive residues, or thefull length of the nucleic acid or polypeptide. The extent of sequenceidentity (homology) may be determined using any computer program andassociated parameters, including those described herein, such as BLAST2.2.2. or FASTA version 3.0t78, with the default parameters.

FIG. 11 is a chart describing selected characteristics of exemplarynucleic acids and polypeptides of the invention, including sequenceidentity comparison of the exemplary sequences to public databases. Allsequences described in FIG. 11 have been subject to a BLAST search (asdescribed in detail, below) against two sets of databases. The firstdatabase set is available through NCBI (National Center forBiotechnology Information). All results from searches against thesedatabases are found in the columns entitled “NR Description”, “NRAccession Code”, “NR Evalue” or “NR Organism”. “NR” refers to theNon-Redundant nucleotide database maintained by NCBI. This database is acomposite of GenBank, GenBank updates, and EMBL updates. The entries inthe column “NR Description” refer to the definition line in any givenNCBI record, which includes a description of the sequence, such as thesource organism, gene name/protein name, or some description of thefunction of the sequence. The entries in the column “NR Accession Code”refer to the unique identifier given to a sequence record. The entriesin the column “NR Evalue” refer to the Expect value (Evalue), whichrepresents the probability that an alignment score as good as the onefound between the query sequence (the sequences of the invention) and adatabase sequence would be found in the same number of comparisonsbetween random sequences as was done in the present BLAST search. Theentries in the column “NR Organism” refer to the source organism of thesequence identified as the closest BLAST hit. The second set ofdatabases is collectively known as the Geneseq™ database, which isavailable through Thomson Derwent (Philadelphia, Pa.). All results fromsearches against this database are found in the columns entitled“Geneseq Protein Description”, “Geneseq Protein Accession Code”,“Geneseq Protein Evalue”, “Geneseq DNA Description”, “Geneseq DNAAccession Code” or “Geneseq DNA Evalue”. The information found in thesecolumns is comparable to the information found in the NR columnsdescribed above, except that it was derived from BLAST searches againstthe Geneseq database instead of the NCBI databases. In addition, thistable includes the column “Predicted EC No.”. An EC number is the numberassigned to a type of enzyme according to a scheme of standardizedenzyme nomenclature developed by the Enzyme Commission of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). The results in the “Predicted EC No.” columnare determined by a BLAST search against the Kegg (Kyoto Encyclopedia ofGenes and Genomes) database. If the top BLAST match has an Evalue equalto or less than e⁻⁶, the EC number assigned to the top match is enteredinto the table. The EC number of the top hit is used as a guide to whatthe EC number of the sequence of the invention might be. The columns“Query DNA Length” and “Query Protein Length” refer to the number ofnucleotides or the number amino acids, respectively, in the sequence ofthe invention that was searched or queried against either the NCBI orGeneseq databases. The columns “Geneseq or NR DNA Length” and “Geneseqor NR Protein Length” refer to the number of nucleotides or the numberamino acids, respectively, in the sequence of the top match from theBLAST search. The results provided in these columns are from the searchthat returned the lower Evalue, either from the NCBI databases or theGeneseq database. The columns “Geneseq or NR % ID Protein” and “Geneseqor NR % ID DNA” refer to the percent sequence identity between thesequence of the invention and the sequence of the top BLAST match. Theresults provided in these columns are from the search that returned thelower Evalue, either from the NCBI databases or the Geneseq database.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence (an exemplarysequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, etc.) to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are entered into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. Default program parameters can be used, oralternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the testsequences relative to the reference sequence, based on the programparameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary sequence of theinvention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, etc., are compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary sequence of the invention,e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto a sequence of the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8,etc., that sequence is within the scope of the invention. In alternativeembodiments, subsequences ranging from about 20 to 600, about 50 to 200,and about 100 to 150 are compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequence for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search forsimilarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444, 1988, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection. Other algorithms for determininghomology or identity include, for example, in addition to a BLASTprogram (Basic Local Alignment Search Tool at the National Center forBiological Information), ALIGN, AMAS (Analysis of Multiply AlignedSequences), AMPS (Protein Multiple Sequence Alignment), ASSET (AlignedSegment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henilcoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used. default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “−F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the invention, andto determine the values in FIG. 11, as discussed above, include:

-   -   “Filter for low complexity: ON    -   >Word Size: 3    -   >Matrix: Blosum62    -   >Gap Costs: Existence: 11    -   >Extension: 1″        Other default settings are: filter for low complexity OFF, word        size of 3 for protein, BLOSUM62 matrix, gap existence penalty of        −11 and a gap extension penalty of −1.

An exemplary NCBI BLAST 2.2.2 program setting is set forth in Example 1,below. Note that the “−W” option defaults to 0. This means that, if notset, the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a polypeptide or nucleic acid sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer. Accordingly, the inventionprovides computers, computer systems, computer readable mediums,computer programs products and the like recorded or stored thereon thenucleic acid and polypeptide sequences of the invention, e.g., anexemplary sequence of the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, etc. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any known methods for recording information on acomputer readable medium to generate manufactures comprising one or moreof the nucleic acid and/or polypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media,magnetic/optical media, flash memories. For example, the computerreadable media may be a hard disk, a floppy disk, a magnetic tape, aflash memory, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory(RAM), or Read Only Memory (ROM), or any type of media known to thoseskilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some embodiments, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100. Software for accessing and processing the nucleotide oramino acid sequences of the invention can reside in main memory 115during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of an exemplary sequence, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, etc. stored on a computer readable medium to reference sequencesstored on a computer readable medium to identify homologies orstructural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention and a sequence comparer for conducting thecomparison. The sequence comparer may indicate a homology level betweenthe sequences compared or identify structural motifs, or it may identifystructural motifs in sequences which are compared to these nucleic acidcodes and polypeptide codes.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 ina computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with a every character in a second sequence,the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention may be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171 or SEQ ID NO:173, or a nucleic acid that encodes a polypeptidecomprising a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,or SEQ ID NO:174. The stringent conditions can be highly stringentconditions, medium stringent conditions, low stringent conditions,including the high and reduced stringency conditions described herein.In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of the molecule, e.g., anexemplary nucleic acid of the invention. For example, they can be atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90,100, 150, 200, 250, 300, 350, 400 or more residues in length. Nucleicacids shorter than full length are also included. These nucleic acidsare useful as, e.g., hybridization probes, labeling probes, PCRoligonucleotide probes, iRNA (single or double stranded), antisense orsequences encoding antibody binding peptides (epitopes), motifs, activesites, binding domains, regulatory domains and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C. Alternatively, nucleic acids of the invention aredefined by their ability to hybridize under high stringency comprisingconditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and arepetitive sequence blocking nucleic acid, such as cot-1 or salmon spermDNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA). In oneaspect, nucleic acids of the invention are defined by their ability tohybridize under reduced stringency conditions comprising 35% formamideat a reduced temperature of 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionscan be used to practice the invention and are well known in the art.Hybridization conditions are discussed further, below.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes for identifying nucleicacids encoding a polypeptide having a phospholipase activity. In oneaspect, the probe comprises at least 10 consecutive bases of a sequenceas set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 or SEQ ID NO:173.Alternatively, a probe of the invention can be at least about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 150, or more, orabout 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of asequence as set forth in a sequence of the invention. The probesidentify a nucleic acid by binding or hybridization. The probes can beused in arrays of the invention, see discussion below, including, e.g.,capillary arrays. The probes of the invention can also be used toisolate other nucleic acids or polypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequencespresent in the sample. Where necessary, conditions which permit theprobe to specifically hybridize to complementary sequences may bedetermined by placing the probe in contact with complementary sequencesfrom samples known to contain the complementary sequence, as well ascontrol sequences which do not contain the complementary sequence.Hybridization conditions, such as the salt concentration of thehybridization buffer, the formamide concentration of the hybridizationbuffer, or the hybridization temperature, may be varied to identifyconditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH2PO₄, pH 7.0, 5.0 mMNa2EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×107 cpm (specific activity 4−9×108 cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is the length of theprobe. Prehybridization may be carried out in 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA or6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmentedsalmon sperm DNA, 50% formamide. Formulas for SSC and Denhardt's andother solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used topractice the invention, e.g., to wash filters. One of skill in the artwould know that there are numerous recipes for different stringencywashes, all of which can be used to practice the invention.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 99%, at least 98%, at least97%, at least 96%, at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 65%, at least 60%, at least55%, or at least 50% homology to a nucleic acid sequence of theinvention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 250, 300, 350, 400, or 500 consecutive bases thereof, andthe sequences complementary thereto. Homology may be measured using analignment algorithm, as discussed herein. For example, the homologouspolynucleotides may have a coding sequence which is a naturallyoccurring allelic variant of one of the coding sequences describedherein. Such allelic variants may have a substitution, deletion oraddition of one or more nucleotides when compared to nucleic acids ofthe invention.

Additionally, the probes and methods of the invention may be used toisolate nucleic acids which encode polypeptides having at least about99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least50% sequence identity (homology) to a polypeptide of the inventioncomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof as determined using a sequence alignmentalgorithm (e.g., such as the FASTA version 3.0t78 algorithm with thedefault parameters, or a BLAST 2.2.2 program with exemplary settings asset forth herein).

Inhibiting Expression of Phospholipases

The invention further provides for nucleic acids complementary to (e.g.,antisense sequences to) the nucleic acids of the invention, e.g.,phospholipase-encoding nucleic acids. Antisense sequences are capable ofinhibiting the transport, splicing or transcription ofphospholipase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA. The transcription or functionof targeted nucleic acid can be inhibited, for example, by hybridizationand/or cleavage. One particularly useful set of inhibitors provided bythe present invention includes oligonucleotides which are able to eitherbind phospholipase gene or message, in either case preventing orinhibiting the production or function of phospholipase enzyme. Theassociation can be though sequence specific hybridization. Anotheruseful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of phospholipase message. The oligonucleotidecan have enzyme activity which causes such cleavage, such as ribozymes.The oligonucleotide can be chemically modified or conjugated to anenzyme or composition capable of cleaving the complementary nucleicacid. One may screen a pool of many different such oligonucleotides forthose with the desired activity.

Inhibition of phospholipase expression can have a variety of industrialapplications. For example, inhibition of phospholipase expression canslow or prevent spoilage. Spoilage can occur when lipids orpolypeptides, e.g., structural lipids or polypeptides, are enzymaticallydegraded. This can lead to the deterioration, or rot, of fruits andvegetables. In one aspect, use of compositions of the invention thatinhibit the expression and/or activity of phospholipase, e.g.,antibodies, antisense oligonucleotides, ribozymes and RNAi, are used toslow or prevent spoilage. Thus, in one aspect, the invention providesmethods and compositions comprising application onto a plant or plantproduct (e.g., a fruit, seed, root, leaf, etc.) antibodies, antisenseoligonucleotides, ribozymes and RNAi of the invention to slow or preventspoilage. These compositions also can be expressed by the plant (e.g., atransgenic plant) or another organism (e.g., a bacterium or othermicroorganism transformed with a phospholipase gene of the invention).

The compositions of the invention for the inhibition of phospholipaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingphospholipase message which can inhibit phospholipase activity bytargeting mRNA. Strategies for designing antisense oligonucleotides arewell described in the scientific and patent literature, and the skilledartisan can design such phospholipase oligonucleotides using the novelreagents of the invention. For example, gene waking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensephospholipase sequences of the invention (see, e.g., Gold (1995) J. ofBiol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides for with ribozymes capable of bindingphospholipase message which can inhibit phospholipase enzyme activity bytargeting mRNA. Strategies for designing ribozymes and selecting thephospholipase-specific antisense sequence for targeting are welldescribed in the scientific and patent literature, and the skilledartisan can design such ribozymes using the novel reagents of theinvention. Ribozymes act by binding to a target RNA through the targetRNA binding portion of a ribozyme which is held in close proximity to anenzymatic portion of the RNA that cleaves the target RNA. Thus, theribozyme recognizes and binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif,but may also be formed in the motif of a hairpin, hepatitis delta virus,group I intron or RNaseP-like RNA (in association with an RNA guidesequence). Examples of such hammerhead motifs are described by Rossi(1992) Aids Research and Human Retroviruses 8:183; hairpin motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a phospholipase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a phospholipase gene. Inone aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or more duplex nucleotides in length. While the invention is not limitedby any particular mechanism of action, the RNAi can enter a cell andcause the degradation of a single-stranded RNA (ssRNA) of similar oridentical sequences, including endogenous mRNAs. When a cell is exposedto double-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. No. 6,506,559; U.S. Pat. No.6,511,824; U.S. Pat. No. 6,515,109; U.S. Pat. No. 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a phospholipase enzyme. Inalternative embodiment, the invention provides methods for modifying anenzyme of the invention, e.g., by mutation of its coding sequence byrandom or stochastic methods, or, non-stochastic, or “directedevolution,” such as Gene Site Saturation Mutagenesis™ (GSSM™), to alterthe enzymes pH range of activity or range of optimal activity,temperature range of activity or range of optimal activity, specificity,activity (kinetics); the enzyme's use of glycosylation, phosphorylationor metals (e.g., Ca, Mg, Zn, Fe, Na), e.g., to impact pH/temperaturestability. The invention provides methods for modifying an enzyme of theinvention, e.g., by mutation of its coding sequence, e.g., by GSSM™, toincrease its resistance to protease activity. The invention providesmethods for modifying an enzyme of the invention, e.g., by mutation ofits coding sequence, e.g., by GSSM™, to modify the enzyme's use of metalchelators specific for Ca, Mg, Na that would not chelate Zn. Theinvention provides methods for modifying an enzyme of the invention,e.g., by mutation of its coding sequence, e.g., by GSSM™, that wouldhave a desired combination of activities, e.g., PI, PA and PC/PEspecific PLCs.

These methods can be repeated or used in various combinations togenerate phospholipase enzymes having an altered or different activityor an altered or different stability from that of a phospholipaseencoded by the template nucleic acid. These methods also can be repeatedor used in various combinations, e.g., to generate variations ingene/message expression, message translation or message stability. Inanother aspect, the genetic composition of a cell is altered by, e.g.,modification of a homologous gene ex vivo, followed by its reinsertioninto the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods.

Methods for random mutation of genes are well known in the art, see,e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be used torandomly mutate a gene. Mutagens include, e.g., ultraviolet light orgamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid,photoactivated psoralens, alone or in combination, to induce DNA breaksamenable to repair by recombination. Other chemical mutagens include,for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine orformic acid. Other mutagens are analogues of nucleotide precursors,e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or combinatorial multiple cassette mutagenesis, see, e.g.,Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleic acids,e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis™ (GSSM™), synthetic ligation reassembly(SLR), recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation, and/or a combination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include pointmismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887),mutagenesis using repair-deficient host strains (Carter et al. (1985)“Improved oligonucleotide site-directed mutagenesis using M13 vectors”Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Certain U.S. applications provide additional details regarding variousdiversity generating methods, including “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No. 09/407,800);“EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCERECOMBINATION” by del Cardayre et al., filed Jul. 15, 1998 (U.S. Ser.No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18,2000 (PCT/US00/01203); “USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESISFOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser.No. 09/408,393); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES& POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al.,filed Jan. 18, 2000, (PCT/US00/01202) and, e.g. “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.09/618,579); “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000(PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affliolter, filedSep. 6, 2000 (U.S. Ser. No. 09/656,549).

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (e.g., GSSM™), synthetic ligation reassembly(SLR), or a combination thereof are used to modify the nucleic acids ofthe invention to generate phospholipases with new or altered properties(e.g., activity under highly acidic or alkaline conditions, hightemperatures, and the like). Polypeptides encoded by the modifiednucleic acids can be screened for an activity before testing for aphospholipase or other activity. Any testing modality or protocol can beused, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM™

In one aspect of the invention, non-stochastic gene modification, a“directed evolution process,” is used to generate phospholipases withnew or altered properties. Variations of this method have been termed“gene site mutagenesis,” “site-saturation mutagenesis,” “Gene SiteSaturation Mutagenesis™” or simply “GSSM™.” It can be used incombination with other mutagenization processes. See, e.g., U.S. Pat.Nos. 6,171,820; 6,238,884. In one aspect, GSSM™ comprises providing atemplate polynucleotide and a plurality of oligonucleotides, whereineach oligonucleotide comprises a sequence homologous to the templatepolynucleotide, thereby targeting a specific sequence of the templatepolynucleotide, and a sequence that is a variant of the homologous gene;generating progeny polynucleotides comprising non-stochastic sequencevariations by replicating the template polynucleotide with theoligonucleotides, thereby generating polynucleotides comprisinghomologous gene sequence variations.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, so as togenerate a set of progeny polypeptides in which a full range of singleamino acid substitutions is represented at each amino acid position,e.g., an amino acid residue in an enzyme active site or ligand bindingsite targeted to be modified. These oligonucleotides can comprise acontiguous first homologous sequence, a degenerate N,N,G/T sequence,and, optionally, a second homologous sequence. The downstream progenytranslational products from the use of such oligonucleotides include allpossible amino acid changes at each amino acid site along thepolypeptide, because the degeneracy of the N,N,G/T sequence includescodons for all 20 amino acids. In one aspect, one such degenerateoligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) isused for subjecting each original codon in a parental polynucleotidetemplate to a full range of codon substitutions. In another aspect, atleast two degenerate cassettes are used—either in the sameoligonucleotide or not, for subjecting at least two original codons in aparental polynucleotide template to a full range of codon substitutions.For example, more than one N,N,G/T sequence can be contained in oneoligonucleotide to introduce amino acid mutations at more than one site.This plurality of N,N,G/T sequences can be directly contiguous, orseparated by one or more additional nucleotide sequence(s). In anotheraspect, oligonucleotides serviceable for introducing additions anddeletions can be used either alone or in combination with the codonscontaining an N,N,G/T sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,phospholipase) molecules such that all 20 natural amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide (other aspectsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g. cloned into asuitable host, e.g., E. coli host, using, e.g., an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedphospholipase activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate phospholipases with new or altered properties. SLRis a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. patent application Ser. No.09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10100 different chimeras. SLR can be used to generatelibraries comprised of over 101000 different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled.

In one aspect of this method, the sequences of a plurality of parentalnucleic acid templates are aligned in order to select one or moredemarcation points. The demarcation points can be located at an area ofhomology, and are comprised of one or more nucleotides. Thesedemarcation points are preferably shared by at least two of theprogenitor templates. The demarcation points can thereby be used todelineate the boundaries of oligonucleotide building blocks to begenerated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in anotherembodiment, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic) asdescribed above. Because of the non-stochastic nature of this invention,the possibility of unwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecularly homologousdemarcation points and thus to allow an increased number of couplings tobe achieved among the building blocks, which in turn allows a greaternumber of progeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate phospholipases withnew or altered properties. Optimized directed evolution is directed tothe use of repeated cycles of reductive reassortment, recombination andselection that allow for the directed molecular evolution of nucleicacids through recombination. Optimized directed evolution allowsgeneration of a large population of evolved chimeric sequences, whereinthe generated population is significantly enriched for sequences thathave a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 1013 chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found in U.S. Ser. No.09/332,835. The number of oligonucleotides generated for each parentalvariant bears a relationship to the total number of resulting crossoversin the chimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding an polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 1013 chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that aoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. One can calculate such a probability density function,and thus enrich the chimeric progeny population for a predeterminednumber of crossover events resulting from a particular ligationreaction. Moreover, a target number of crossover events can bepredetermined, and the system then programmed to calculate the startingquantities of each parental oligonucleotide during each step in theligation reaction to result in a probability density function thatcenters on the predetermined number of crossover events.

Determining Crossover Events

Embodiments of the invention include a system and software that receivea desired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB® (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example a nucleic acid (or, the nucleic acid) responsiblefor an altered phospholipase phenotype is identified, re-isolated, againmodified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including phospholipase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new phospholipasephenotype), it can be removed as a variable by synthesizing largerparental oligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,phospholipase enzymes, and the like. In vivo shuffling can be performedutilizing the natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides methods of making sequence variants of thenucleic acid and phospholipase sequences of the invention or isolatingphospholipase enzyme, e.g., phospholipase, sequence variants using thenucleic acids and polypeptides of the invention. In one aspect, theinvention provides for variants of a phospholipase gene of theinvention, which can be altered by any means, including, e.g., random orstochastic methods, or, non-stochastic, or “directed evolution,”methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In someembodiments, random mutations in a sequence of interest are generated bypropagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some embodiments, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250.

The invention also provides variants of polypeptides of the inventioncomprising sequences in which one or more of the amino acid residues(e.g., of an exemplary polypeptide of the invention) are substitutedwith a conserved or non-conserved amino acid residue (e.g., a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code. Conservative substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Thus, polypeptides of the inventioninclude those with conservative substitutions of sequences of theinvention, including but not limited to the following replacements:replacements of an aliphatic amino acid such as Alanine, Valine, Leucineand Isoleucine with another aliphatic amino acid; replacement of aSerine with a Threonine or vice versa; replacement of an acidic residuesuch as Aspartic acid and Glutamic acid with another acidic residue;replacement of a residue bearing an amide group, such as Asparagine andGlutamine, with another residue bearing an amide group; exchange of abasic residue such as Lysine and Argmine with another basic residue; andreplacement of an aromatic residue such as Phenylalanine, Tyrosine withanother aromatic residue. Other variants are those in which one or moreof the amino acid residues of the polypeptides of the invention includesa substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., a phospholipase activity,as described herein. In other aspects, the variant, fragment,derivative, or analog includes a proprotein, such that the variant,fragment, derivative, or analog can be activated by cleavage of theproprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying phospholipase-encodingnucleic acids to modify codon usage. In one aspect, the inventionprovides methods for modifying codons in a nucleic acid encoding aphospholipase to increase or decrease its expression in a host cell. Theinvention also provides nucleic acids encoding a phospholipase modifiedto increase its expression in a host cell, phospholipase enzymes somodified, and methods of making the modified phospholipase enzymes. Themethod comprises identifying a “non-preferred” or a “less preferred”codon in phospholipase-encoding nucleic acid and replacing one or moreof these non-preferred or less preferred codons with a “preferred codon”encoding the same amino acid as the replaced codon and at least onenon-preferred or less preferred codon in the nucleic acid has beenreplaced by a preferred codon encoding the same amino acid. A preferredcodon is a codon over-represented in coding sequences in genes in thehost cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as any Bacillus (e.g., B. cereus) orStreptomyces, Lactobacillus gasseri, Lactococcus lactis, Lactococcuscremoris, Bacillus subtilis. Exemplary host cells also includeeukaryotic organisms, e.g., various yeast, such as Saccharoinyces sp.,including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillusniger, and mammalian cells and cell lines and insect cells and celllines. Thus, the invention also includes nucleic acids and polypeptidesoptimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding a phospholipaseisolated from a bacterial cell are modified such that the nucleic acidis optimally expressed in a bacterial cell different from the bacteriafrom which the phospholipase was derived, a yeast, a fungi, a plantcell, an insect cell or a mammalian cell. Methods for optimizing codonsare well known in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca(2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif.12:185-188; Narum (2001) Infect. Immun. 69:7250-7253. See also Narum(2001) Infect. Immun. 69:7250-7253, describing optimizing codons inmouse systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,describing optimizing codons in yeast; Feng (2000) Biochemistry39:15399-15409, describing optimizing codons in E. coli; Humphreys(2000) Protein Expr. Purif. 20:252-264, describing optimizing codonusage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The transgenic non-human animalscan be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice,comprising the nucleic acids of the invention. These animals can beused, e.g., as in vivo models to study phospholipase activity, or, asmodels to screen for modulators of phospholipase activity in vivo. Thecoding sequences for the polypeptides to be expressed in the transgenicnon-human animals can be designed to be constitutive, or, under thecontrol of tissue-specific, developmental-specific or inducibletranscriptional regulatory factors. Transgenic non-human animals can bedesigned and generated using any method known in the art; see, e.g.,U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usingtransformed cells and eggs and transgenic mice, rats, rabbits, sheep,pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods231:147-157, describing the production of recombinant proteins in themilk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol.17:456-461, demonstrating the production of transgenic goats. U.S. Pat.No. 6,211,428, describes making and using transgenic non-human mammalswhich express in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice whose cells express proteins related to thepathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express or to be unable to express aphospholipase.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a phospholipase), an expression cassette orvector or a transfected or transformed cell of the invention. Theinvention also provides plant products, e.g., oils, seeds, leaves,extracts and the like, comprising a nucleic acid and/or a polypeptide(e.g., a phospholipase) of the invention. The transgenic plant can bedicotyledonous (a dicot) or monocotyledonous (a monocot). The inventionalso provides methods of making and using these transgenic plants andseeds. The transgenic plant or plant cell expressing a polypeptide ofthe invention may be constructed in accordance with any method known inthe art. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's phospholipase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on oil-seed containing plants, such asrice, soybeans, rapeseed, sunflower seeds, sesame and peanuts. Nucleicacids of the invention can be used to manipulate metabolic pathways of aplant in order to optimize or alter host's expression of phospholipase.The can change phospholipase activity in a plant. Alternatively, aphospholipase of the invention can be used in production of a transgenicplant to produce a compound not naturally produced by that plant. Thiscan lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with nucleicacids, e.g., an expression construct. Although plant regeneration fromprotoplasts is not easy with cereals, plant regeneration is possible inlegumes using somatic embryogenesis from protoplast derived callus.Organized tissues can be transformed with naked DNA using gene guntechnique, where DNA is coated on tungsten microprojectiles, shot1/100th the size of cells, which carry the DNA deep into cells andorganelles. Transformed tissue is then induced to regenerate, usually bysomatic embryogenesis. This technique has been successful in severalcereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., aphospholipase) of the invention. The desired effects can be passed tofuture plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants (e.g., as transgenic plants), such as oil-seedcontaining plants, e.g., rice, soybeans, rapeseed, sunflower seeds,sesame and peanuts. The nucleic acids of the invention can be expressedin plants which contain fiber cells, including, e.g., cotton, silkcotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., a phospholipase orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agiobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

The invention provides isolated or recombinant polypeptides having asequence identity (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or complete (100%) sequence identity) to an exemplarysequence of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108 SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQID NO:136, SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144;NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164,SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, or SEQ IDNO:174. As discussed above, the identity can be over the full length ofthe polypeptide, or, the identity can be over a subsequence thereof,e.g., a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.Polypeptides of the invention can also be shorter than the full lengthof exemplary polypeptides (e.g., SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6;SEQ ID NO:8, etc.). In alternative embodiment, the invention providespolypeptides (peptides, fragments) ranging in size between about 5 andthe full length of a polypeptide, e.g., an enzyme, such as aphospholipase, e.g., phospholipase; exemplary sizes being of about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400 or more residues, e.g.,contiguous residues of the exemplary phospholipases of SEQ ID NO:2; SEQID NO:4; SEQ ID NO:6; SEQ ID NO:8, etc. Peptides of the invention can beuseful as, e.g., labeling probes, antigens, toleragens, motifs,phospholipase active sites, binding domains, regulatory domains, and thelike.

In one aspect, the polypeptide has a phospholipase activity, e.g.,cleavage of a glycerolphosphate ester linkage, the ability to hydrolyzephosphate ester bonds, including patatin, lipid acyl hydrolase (LAH),phospholipase A, B, C and/or phospholipase D activity, or anycombination thereof.

In alternative aspects, exemplary polypeptides of the invention have aphospholipase activity, Signal Sequence Location, and an initial source,as set forth in the following Table 1, below. To aid in reading thetable, for example, in the first row, where SEQ ID NO:143, 144, meansthe polypeptide having a sequence as set forth in SEQ ID NO:144, andencoded by, e.g., SEQ ID NO:143, having a PLA-specific PLA activity,initially isolated from an unknown source; another example in the SEQ IDNO:167, 168 row where 167, 168 means the polypeptide having a sequenceas set forth in SEQ ID NO:168, and encoded by, e.g., SEQ ID NO:167,having a phosphatidic acid phosphatase activity, a signal sequence atresidues 1 to 30 (“AA1-30” means amino acid residues 1 to 30, etc.),i.e., MARSWKWRPLLSSFLLVSLAPFSTSVPCFK, and initially isolated from anunknown source. The invention also provides peptides comprising signalsequences, and chimeric polypeptides, where the peptides or chimericscomprise signal sequences as set forth in Table 1, and as describedbelow.

TABLE 1 Signal Seq. Location (AA = Amino SEQ ID NO: Enzyme type Acid)Signal (AA) Source 143, PA-specific 144 PLA Unknown 25, 26 PatatinUnknown 77, 78 Patatin Unknown 35, 36 Patatin Unknown 125, PatatinUnknown 126 135, 136 Patatin Unknown 99, 100 Patatin Unknown 65, 66Patatin Unknown 87, 88 Patatin Unknown 86, 87 Patatin Unknown 45, 46Patatin Unknown 59, 60 Patatin Unknown 13, 14 Patatin Unknown 71, 72Patatin Unknown 55, 56 Patatin Unknown 33, 34 Patatin Unknown 91, 92Patatin Unknown 103 104 Patatin Unknown 11, 12 Patatin Unknown 17, 18Patatin Unknown 95, 96 Patatin Unknown 43, 44 Patatin Unknown 27, 28Patatin Unknown 131, 132 Patatin Unknown 127, 128 Patatin Unknown 133,134 Patatin Unknown 137, 138 Patatin Unknown 165, 166 Patatin UnknownPbosphatidic 167, acid MARSWKWRPLLSSFL 168 phosphatases AA1-30LVSLAPFSTSVPCFK Unknown Phosphatidic 169, acid 170 phosphatases UnknownPhosphatidic 171, acid 172 phosphatases Unknown Phosphatidic 173, acid174 phosphatases Unknown 111, Phosphatidy 112 linositol PLC AA1-16MGAGAILLTGAPTASA Bacteria 107, Phosphatidy- 108 linositol PLC AA1-23MSNKKFILKLFICSTI Unknown LSTFVFA 109, Phosphatidy 110 linositol PLCAA1-23 MSNKKFILKLFICSTI Unknown LSTFVFA 113 phosphatidylin 114 ositolPLC AA1-23 MSNKKFILKLFICSTI Unknown LSTFVFA 117, Phosphatidy- 118linositol PLC AA1-23 MNNKKFILKLFICSMV Unknown LSAFVFA 119,phosphatidylin 120 ositol PLC AA1-23 MNNKKFILKLFICSMV Unknown LSAFVFA115 Phosphatidy- 116 linositol PLC AA1-23 MNNKKFILKLFICSMV UnknownLSAFVFA 121, Phosphatidy- 122 linositol PLC AA1-23 MRNKKFILKLLICSTVUnknown LSTFVFA 141, 142 Phospholipase Unknown 155, MRTTTTNWRQIVKSLK 156Phospholipase AA1-36 LFLMGLCLFISASSAY Unknown A 159, 160 PbospholipaseUnknown 145, 146 PLA Unknown 147, 148 PLA Unknown 149, 150 PLA Unknown151, 152 PLA Unknown 153, 154 PLA Unknown 157, 158 PLA Unknown 163, 164PLA Unknown 101, LSLVASLRRAPGAALA 102 PLC AA1-39 LALAAATLAVTAQGATBacteria AAPAAAAA 1, 2 PLC AA1-24 MKKKVLALAAMVALAA Unknown PVQSWFAQ 3, 4PLC AA1-24 MKRKILAIASVIALTA Unknown PIQSVAFAH 5, 6 PLC AA1-24MKRKILAIASVIALTA Unknown PIQSVAFAH 97, 98 PLC AA1-25 MKRKLCTWALVTAIASUnknown STAVIPTAAE 7, 8 PLC AA1-29 MITLIKKCLLVLTMTL UnknownLLGVFVPLQPSHAT 31, 32 PLC AA1-20 MKKKLCTWALVTAISS Unknown GVVAI 81, 82PLC AA1-25 MKKKLCTMALVTAISS Unknown GVVTIPTEAQ 93, 94 PLC AA1-29MITLIKKCLLVLTMTL Unknown LSGVFVPLQPSYAT 89, 90 PLC AA1-25MKKKLCTLAFVTAISS Unknown IAITIPTEAQ 123, 124 PLC AA1-24 MKKKVLALAAMVALAAUnknown PVQSWFA 129, 130 PLC AA1-27 MKKKICTLALVSAITS Unknown GVVTIPTVASA139, 140 PLC AA1-20 MKIKPLTFSFGLAVTS Unknown SVQA 105, MNRCRNSLNLQLRAVT106 PLC AA1-30 VAALVVVASSAALAW Unknown 9, 10 PLC AA1-20 MKLLRVFVCVFALLSAUnknown HSKAD 47, 48 PLD Unknown 15, 16 PLD Unknown 41, 42 PLD Unknown23, 24 PLD Unknown 51, 52 PLD Unknown 53, 54 PLD Unknown 19, 20 PLDAA1-19 MKKTTLVLALLMPFGA Unknown ASAQ 75, 76 PLD Unknown 57, 58 PLDUnknown 63, 64 PLD AA1-18 MKNTLILAGCILAAPA Unknown VAD 79, 80 PLD AA1-23MRNFSKGLTSILLSIA Unknown TSTSAMAF 37, 38 PLD AA1-23 MRNFSKGLTSILLSIAUnknown TSTSAMAF 61, 62 PLD AA1-21 MTLKLSLLIASLSAVS Unknown PAVLAN 67,68 PLD No Unknown 83, 84 PLD AA1-21 MKKIVIYSFVAGVMTS Unknown GGVFAA 49,50 PLD AA1-23 MNFWSFLLSITLPMGV Unknown GVAHAQPD 39, 40 PLD Unknown 73,74 PLD Unknown 29, 30 PLD Unknown 21, 22 PLD AA1-28 MQQHKLRNFNKGLTGVUnknown VLSVLTSTSAMAF 71, 72 PLD Unknown 161, 162 PLD AA1-24MNRKLLSLCLGATSCI Unknown ALSLPVHA

In one aspect, the invention provides polypeptides having sequences asset forth in SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113,SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ IDNO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141,SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ IDNO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169,SEQ ID NO:171 and/or SEQ ID NO:173, and subsequences thereof, e.g.,their active sites (“catalytic domains”) having a phospholipaseactivity, e.g., a phospholipase C (PLC) activity. In one aspect, thepolypeptide has a phospholipase activity but lacks neutral oil(triglyceride) hydrolysis activity. For example, in one aspect, thepolypeptide has a phospholipase activity but lacks any activity thataffects a neutral oil (triglyceride) fraction. In one aspect, theinvention provides a degumming process comprising use of a polypeptideof the invention having a phospholipase activity, but not a lipaseactivity.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has aphospholipase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-di-isopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH2 for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Delker, N.Y.).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenyl-phenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole-(alkyl)alanines;and, D- or L-alkylainines, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromaticrings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl,pyrazolyl, benznimdazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where allyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R— or S— form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

Phospholipase Enzymes

The invention provides novel phospholipases, nucleic acids encodingthem, antibodies that bind them, peptides representing the enzyme'santigenic sites (epitopes) and active sites, regulatory and bindingdomains, and methods for making and using them. In one aspect,polypeptides of the invention have a phospholipase activity, or anycombination of phospholipase activities, as described herein (e.g.,cleavage of a glycerolphosphate ester linkage, lacking lipase activity,etc.). In alternative aspects, the phospholipases of the invention haveactivities that have been modified from those of the exemplaryphospholipases described herein.

The invention includes phospholipases with and without signal sequencesand the signal sequences themselves. The invention includes fragments orsubsequences of enzymes of the invention, e.g., peptides or polypeptidescomprising or consisting of catalytic domains (“active sites”), bindingsites, regulatory domains, epitopes, signal sequences, prepro domains,and the like. The invention also includes immobilized phospholipases,anti-phospholipase antibodies and fragments thereof. The inventionincludes heterocomplexes, e.g., fusion proteins, heterodimers, etc.,comprising the phospholipases of the invention. Determining peptidesrepresenting the enzyme's antigenic sites (epitopes), active sites,binding sites, signal sequences, and the like can be done by routinescreening protocols.

These enzymes and processes of the invention can be used to achieve amore complete degumming of high phosphorus oils, in particular, rice,soybean, corn, canola, and sunflower oils. For example, in one aspect,upon cleavage by PI-PLC, phosphatidylinositol is converted todiacylglycerol and phosphoinositol. The diacylglycerol partitions to theaqueous phase (improving oil yield) and the phosphoinositol partitionsto the aqueous phase where it is removed as a component of the heavyphase during centrifugation. An enzyme of the invention, e.g., a PI-PLCof the invention, can be incorporated into either a chemical or physicaloil refining process.

In alternative aspects, enzymes of the invention havephosphatidylinositol-specific phospholipase C (PI-PLC) activity,phosphatidylcholine-specific phospholipase C activity, phosphatidic acidphosphatase activity, phospholipase A activity and/or patatin-relatedphospholipase activity. These enzymes can be used alone or incombination each other or with other enzymes of the invention, or otherenzymes. In one aspect, the invention provides methods wherein theseenzymes (including phosphatidylinositol-specific phospholipase C(PIPLC), phosphatidylcholine-specific phospholipase C, and/orphospholipase D (in conjunction with a phosphatase), phosphatidic acidphosphatase, phospholipase A, patatin-related phospholipases of theinvention) are used alone or in combination in the degumming of oils,e.g., vegetable oils, e.g., high phosphorus oils, such as soybean, corn,canola, rice bran and sunflower oils. These enzymes and processes of theinvention can be used to achieve a more complete degumming of highphosphorus oils, in particular, soybean, corn, canola, rice bran andsunflower oils. Upon cleavage by PI-PLC, phosphatidylinositol isconverted to diacylglycerol and phosphoinositol. The diacylglycerolpartitions to the aqueous phase (improving oil yield) and thephosphoinositol partitions to the aqueous phase where it is removed as acomponent of the heavy phase during centrifugation. An enzyme of theinvention, e.g., a PI-PLC of the invention, can be incorporated intoeither a chemical or physical oil refining process.

In one aspect, the invention provides compositions, e.g., solutions,comprising sodium citrate at neutral pH to hydrate non-hydratables. Forexample, the invention provides sodium citrate solutions in a pH rangeof between about 4 to 9, or, 5 to 8, or, 6 to 7, that can be used tohydrate non-hydratable phospholipids (including enzymes of theinvention) in high phosphorus oils. In one aspect, the hydration ofnon-hydratable phospholipids is by chelating the calcium and magnesiumassociated with the phospholipids, thereby allowing the formerlyinsoluble phospholipid salts to more readily partition in the aqueousphase. In one aspect, once phospholipids move to the water/oil interfaceor into the aqueous phase, a phospholipase of the invention (e.g., aphospholipase-specific phosphohydrolase of the invention), or anotherphospholipase, will convert the phospholipid to diacylglycerol and aphosphate-ester. In one aspect, calcium and magnesium metal content arelowered upon addition of acid and caustic (see discussion on causticprocesses).

The enzymes of the invention are highly selective catalysts. As withother enzymes, they catalyze reactions with exquisite stereo-, region-,and chemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, the enzymes of the invention are remarkablyversatile. They can be tailored to function in organic solvents, operateat extreme pHs (for example, high pHs and low pHs) extreme temperatures(for example, high temperatures and low temperatures), extreme salinitylevels (for example, high salinity and low salinity), and catalyzereactions with compounds that are structurally unrelated to theirnatural, physiological substrates. Enzymes of the invention can bedesigned to be reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Enzymes of the invention can also be designed to be highlyenantio- and regio-selective. The high degree of functional groupspecificity exhibited by these enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes of the invention can also be designed to catalyze many diversereactions unrelated to their native physiological function in nature.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound. The present invention usesselected biocatalysts, i.e., the enzymes of the invention, and reactionconditions that are specific for functional groups that are present inmany starting compounds. Each biocatalyst is specific for one functionalgroup, or several related functional groups, and can react with manystarting compounds containing this functional group. The biocatalyticreactions produce a population of derivatives from a single startingcompound. These derivatives can be subjected to another round ofbiocatalytic reactions to produce a second population of derivativecompounds. Thousands of variations of the original compound can beproduced with each iteration of biocatalytic derivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process that is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active enzyme withina library. The library is characterized by the series of biocatalyticreactions used to produce it, a so-called “biosynthetic history”.Screening the library for biological activities and tracing thebiosynthetic history identifies the specific reaction sequence producingthe active compound. The reaction sequence is repeated and the structureof the synthesized compound determined. This mode of identification,unlike other synthesis and screening approaches, does not requireimmobilization technologies, and compounds can be synthesized and testedfree in solution using virtually any type of screening assay. It isimportant to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

The invention also provides methods of discovering new phospholipasesusing the nucleic acids, polypeptides and antibodies of the invention.In one aspect, lambda phage libraries are screened for expression-baseddiscovery of phospholipases. Use of lambda phage libraries in screeningallows detection of toxic clones; improved access to substrate; reducedneed for engineering a host, by-passing the potential for any biasresulting from mass excision of the library; and, faster growth at lowclone densities. Screening of lambda phage libraries can be in liquidphase or in solid phase. Screening in liquid phase gives greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility (see discussion of arrays, below). As a result, alibrary of derivative compounds can be produced in a matter of weeks.For further teachings on modification of molecules, including smallmolecules, see PCT/US94/09174.

Phospholipase Signal Sequences

The invention provides phospholipase signal sequences (e.g., signalpeptides (SPs)), e.g., peptides comprising signal sequences and/orchimeric polypeptides, where the peptides or chimerics have a signalsequence as set forth in Table 1, or as set forth, below. The inventionprovides nucleic acids encoding these signal sequences (SPs, e.g., apeptide having a sequence comprising/consisting of amino terminalresidues of a polypeptide of the invention). In one aspect, theinvention provides a signal sequence comprising a peptidecomprising/consisting of a sequence as set forth in residues 1 to 20, 1to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1to 28, 1 to 30, 1 to 31, 1 to 32 or 1 to 33 of a polypeptide of theinvention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108 SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ IDNO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166,SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, or SEQ ID NO:174. Any ofthese peptides can be part of a chimeric protein, e.g., a recombinantprotein. A signal sequence peptide can be matched with another enzyme ofthe invention (e.g., a phospholipase of the invention from which is wasnot derived), or, with another phospholipase, or with any polypeptide,as discussed further, below.

Exemplary signal sequences are set forth in Table 1 and the SEQ IDlisting, e.g., residues 1 to 24 of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6; residues 1 to 29 of SEQ ID NO:8; residues 1 to 20 of SEQ ID NO:10;residues 1 to 19 of SEQ ID NO:20; residues 1 to 28 of SEQ ID NO:22;residues 1 to 20 of SEQ ID NO:32; residues 1 to 23 of SEQ ID NO: 38; seeTable 1 and the SEQ ID listing for other exemplary signal sequences ofthe invention.

In some aspects phospholipases of the invention do not have signalsequences. In one aspect, the invention provides the phospholipases ofthe invention lacking all or part of a signal sequence. In one aspect,the invention provides a nucleic acid sequence encoding a signalsequence from one phospholipase operably linked to a nucleic acidsequence of a different phospholipase or, optionally, a signal sequencefrom a non-phospholipase protein may be desired.

Phospholipase Prepro Domains, Binding Domains and Catalytic Domains

In addition to signal sequences (e.g., signal peptides (SPs)), asdiscussed above, the invention provides prepro domains, binding domains(e.g., substrate binding domain) and catalytic domains (CDs). The SPdomains, binding domains, prepro domains and/or CDs of the invention canbe isolated or recombinant peptides or can be part of a fusion protein,e.g., as a heterologous domain in a chimeric protein. The inventionprovides nucleic acids encoding these catalytic domains (CDs) (e.g.,“active sites”), prepro domains, binding domains and signal sequences(SPs, e.g., a peptide having a sequence comprising/consisting of aminoterminal residues of a polypeptide of the invention).

The phospholipase signal sequences (SPs), binding domains, catalyticdomains (CDs) and/or prepro sequences of the invention can be isolatedpeptides, or, sequences joined to another phospholipase or anon-phospholipase polypeptide, e.g., as a fusion (chimeric) protein. Inone aspect, polypeptides comprising phospholipase signal sequences SPsand/or prepro of the invention comprise sequences heterologous tophospholipases of the invention (e.g., a fusion protein comprising an SPand/or prepro of the invention and sequences from another phospholipaseor a non-phospholipase protein). In one aspect, the invention providesphospholipases of the invention with heterologous CDs, SPs and/or preprosequences, e.g., sequences with a yeast signal sequence. A phospholipaseof the invention can comprise a heterologous CD, SP and/or prepro in avector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs, CDs, and/or prepro sequences of the invention areidentified following identification of novel phospholipase polypeptides.The pathways by which proteins are sorted and transported to theirproper cellular location are often referred to as protein targetingpathways. One of the most important elements in all of these targetingsystems is a short amino acid sequence at the amino terminus of a newlysynthesized polypeptide called the signal sequence. This signal sequencedirects a protein to its appropriate location in the cell and is removedduring transport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length from 13to 45 or more amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel hydrolase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eulcaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

In some aspects, a phospholipase of the invention may not have SPsand/or prepro sequences, and/or catalytic domains (CDs). In one aspect,the invention provides phospholipases lacking all or part of an SP, a CDand/or a prepro domain. In one aspect, the invention provides a nucleicacid sequence encoding a signal sequence (SP), a CD and/or prepro fromone phospholipase operably linked to a nucleic acid sequence of adifferent phospholipase or, optionally, a signal sequence (SPs), a CDand/or prepro domain from a non-phospholipase protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa phospholipase) with an SP, prepro domain and/or CD. The sequence towhich the SP, prepro domain and/or CD are not naturally associated canbe on the SP's, prepro domain and/or CD's amino terminal end, carboxyterminal end, and/or on both ends of the SP and/or CD. In one aspect,the invention provides an isolated or recombinant polypeptide comprising(or consisting of) a polypeptide comprising a signal sequence (SP),prepro domain and/or catalytic domain (CD) of the invention with theproviso that it is not associated with any sequence to which it isnaturally associated (e.g., phospholipase sequence). Similarly in oneaspect, the invention provides isolated or recombinant nucleic acidsencoding these polypeptides. Thus, in one aspect, the isolated orrecombinant nucleic acid of the invention comprises coding sequence fora signal sequence (SP), prepro domain and/or catalytic domain (CD) ofthe invention and a heterologous sequence (i.e., a sequence notnaturally associated with the a signal sequence (SP), prepro domainand/or catalytic domain (CD) of the invention). The heterologoussequence can be on the 3′ terminal end, 5′ terminal end, and/or on bothends of the SP, prepro domain and/or CD coding sequence.

The polypeptides of the invention include phospholipases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude phospholipases inactive for other reasons, e.g., before“activation” by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation, a de-glycosylation, a sulfation, adimerization event, and/or the like. Methods for identifying “prepro”domain sequences, CDs, binding domains and signal sequences are routineand well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136; yeast two-hybrid screenings for identifyingprotein-protein interactions, described e.g., by Miller (2004) MethodsMol. Biol. 261:247-62; Heyninck (2004) Methods Mol. Biol. 282:223-41,U.S. Pat. Nos. 6,617,122; 6,190,874. For example, to identify a preprosequence, the protein is purified from the extracellular space and theN-terminal protein sequence is determined and compared to theunprocessed form.

The polypeptides of the invention can be formulated as a proteinpreparation into any liquid, solid, semi-solid or gel form. For example,a protein preparation of the invention can comprise a formulationcomprising a non-aqueous liquid composition, a cast solid, a powder, alyophilized powder, a granular form, a particulate form, a compressedtablet, a pellet, a pill, a gel form, a hydrogel, a paste, an aerosol, aspray, a lotion or a slurry formulation.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains (CDs) or active sites, ofan enzyme of the invention. In one aspect, the invention providescatalytic domains or active sites as set forth below. In one aspect, theinvention provides a peptide or polypeptide comprising or consisting ofan active site domain as predicted through use of a database such asPfam (which is a large collection of multiple sequence alignments andhidden Markov models covering many common protein families, The Pfamprotein families database, A. Bateman, E. Birney, L. Ceruti, R. Durbin,L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall,and E. L. L. Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002) orequivalent.

The invention provides fusion of N-terminal or C-terminal subsequencesof enzymes of the invention (e.g., signal sequences, prepro sequences)with other polypeptides, active proteins or protein fragments. Theproduction of an enzyme of the invention (e.g., a phospholipase Cenzyme) may also be accomplished by expressing the enzyme as an inactivefusion protein that is later activated by a proteolytic cleavage event(using either an endogenous or exogenous protease activity, e.g.trypsin) that results in the separation of the fusion protein partnerand the mature enzyme, e.g., phospholipase C enzyme. In one aspect, thefusion protein of the invention is expressed from a hybrid nucleotideconstruct that encodes a single open reading frame containing thefollowing elements: the nucleotide sequence for the fusion protein, alinker sequence (defined as a nucleotide sequence that encodes aflexible amino acid sequence that joins two less flexible proteindomains), protease cleavage recognition site, and the mature enzyme(e.g., any enzyme of the invention, e.g., a phospholipase) sequence. Inalternative aspects, the fusion protein can comprise a pectate lyasesequence, a xylanase sequence, a phosphatidic acid phosphatase sequence,or another sequence, e.g., a sequence that has previously been shown tobe over-expressed in a host system of interest.

Any host system can be used (see discussion, above), for example, anybacteria, e.g., a gram positive bacteria, such as Bacillus, or a gramnegative bacteria, such as E. coli, or any yeast, e.g., Pichia pastoris.The arrangement of the nucleotide sequences in the chimeric nucleotideconstruction can be determined based on the protein expression levelsachieved with each fusion construct. Proceeding from the 5′ end of thenucleotide construct to the 3′ prime end of the construct, in oneaspect, the nucleotide sequences is assembled as follows: Signalsequence/fusion protein/linker sequence/protease cleavage recognitionsite/mature enzyme (e.g., any enzyme of the invention, e.g., aphospholipase) or Signal sequence/pro sequence/mature enzyme/linkersequence/fusion protein. The expression of enzyme (e.g., any enzyme ofthe invention, e.g., a phospholipase) as an inactive fusion protein mayimprove the overall expression of the enzyme's sequence, may reduce anypotential toxicity associated with the overproduction of active enzymeand/or may increase the shelf life of enzyme prior to use because enzymewould be inactive until the fusion protein e.g. pectate lyase isseparated from the enzyme, e.g., phospholipase protein.

In various aspects, the invention provides specific formulations for theactivation of phospholipase of the invention expressed as a fusionprotein. In one aspect, the activation of the phospholipase activityinitially expressed as an inactive fusion protein is accomplished usinga proteolytic activity or potentially a proteolytic activity incombination with an amino-terminal or carboxyl-terminal peptidase. Thisactivation event may be accomplished in a variety of ways and at varietyof points in the manufacturing/storage process prior to application inoil degumming. Exemplary processes of the invention include: Cleavage byan endogenous activity expressed by the manufacturing host uponsecretion of the fusion construct into the fermentation media; Cleavageby an endogenous protease activity that is activated or comes in contactwith intracellularly expressed fusion construct upon rupture of the hostcells; Passage of the crude or purified fusion construct over a columnof immobilized protease activity to accomplish cleavage and enzyme(e.g., phospholipase of the invention, e.g., a phospholipase C)activation prior to enzyme formulation; Treatment of the crude orpurified fusion construct with a soluble source of proteolytic activity;Activation of a phospholipase (e.g., a phospholipase of the invention,e.g., a phospholipase C) at the oil refinery using either a soluble orinsoluble source of proteolytic activity immediately prior to use in theprocess; and/or, Activation of the phospholipase (e.g., a phospholipaseof the invention, e.g., a phospholipase C) activity by continuouslycirculating the fusion construct formulation through a column ofimmobilized protease activity at reduced temperature (for example, anybetween about 4° C. and 20° C.). This activation event may beaccomplished prior to delivery to the site of use or it may occuron-site at the oil refinery.

Glycosylation

The peptides and polypeptides of the invention (e.g., hydrolases,antibodies) can also be glycosylated, for example, in one aspect,comprising at least one glycosylation site, e.g., an N-linked orO-linked glycosylation. In one aspect, the polypeptide can beglycosylated after being expressed in a P. pastoris or a S. pombe. Theglycosylation can be added post-translationally either chemically or bycellular biosynthetic mechanisms, wherein the later incorporates the useof known glycosylation motifs, which can be native to the sequence orcan be added as a peptide or added in the nucleic acid coding sequence.

In one aspect, the invention provides a polypeptide comprising anN-linked glycosylated SEQ ID NO:2, as described, e.g., in the followingtable:

Site Amino acid position of number Glycosylation site Lengthglycosylation site 1 Match: NNS Length: 3 Start: 27 Stop: 29 2 Match:NTT Length: 3 Start: 65 Stop: 67 3 Match: NET Length: 3 Start: 72 Stop:74 4 Match: NST Length: 3 Start: 100 Stop: 102 5 Match: NFT Length: 3Start: 168 Stop: 170 6 Match: NLS Length: 3 Start: 171 Stop: 173 7Match: NDT Length: 3 Start: 229 Stop: 231

The full-length SEQ ID NO:2 (which in one aspect is encoded by SEQ IDNO:1) open reading frame encodes seven (7) potential asparagine-linked(N-linked) glycosylation sites. The expression of the wild-type SEQ IDNO:2 open reading frame in a glycosylating host (e.g. Pichia pastoris,Saccharomyces cerevisiae, Schizosaccharomyces pombe, or a mammaliancell) results in the production of a glycosylated SEQ ID NO:2phospholipase enzyme that is essentially inactive due to the presence ofN-linked glycosylation. Enzymatic deglycosylation of the wild-type,glycosylated SEQ ID NO:2 with PNGase F or Endoglycosidase H results inthe activation of the SEQ ID NO:2 activity. In addition, modification ofone or more of the N-linked glycosylation sites through mutagenesis (sothat the site is no longer recognized as an N-linked glycosylation siteand glycosylation no longer occurs at that site) results in theproduction of SEQ ID NO:2 with varying degrees of increased activity.

Mutagenesis of the nucleotide codon encoding the asparagine in SEQ IDNO:2 glycosylation sites 4,5, and/or 6 (e.g. converting the asparagineto an aspartic acid) results in the production of an enzyme withincreased PLC activity compared to the wild-type open reading frameexpressed in the same host (the triple mutant expressed in Pichiapastoris possesses a specific activity and a functional activity that isessentially identical to that of the wild-type sequence expressed in anon-glycosylating host like E. coli. It is also possible to abolish theN-linked glycosylation site by mutagenesis of the serine or threonineresidue in the N-linked glycosylation consensus sequence (NXS/T), forexample by converting these nucleotide codons to produce valine orisoleucine at these positions instead of serine or threonine. The use ofthis strategy to remove N-linked glycosylation sites also results in theproduction of active SEQ ID NO:2 phospholipase in glycosylating hostexpression systems.

Assays for Phospholiase Activity

The invention provides isolated, synthetic or recombinant polypeptides(e.g., enzymes, antibodies) having a phospholipase activity, or anycombination of phospholipase activities, and nucleic acids encodingthem. Any of the many phospholipase activity assays known in the art canbe used to determine if a polypeptide has a phospholipase activity andis within the scope of the invention. Routine protocols for determiningphospholipase A, B, D and C, patatin and lipid acyl hydrolaseactivities, or lipase activity, are well known in the art.

Exemplary activity assays include turbidity assays, methylumbelliferylphosphocholine (fluorescent) assays, Amplex red (fluorescent)phospholipase assays, thin layer chromatography assays (TLC), cytolyticassays and p-nitrophenylphosphorylcholine assays. Using these assayspolypeptides, peptides or antibodies can be quickly screened for aphospholipase activity.

The phospholipase activity can comprise a lipid acyl hydrolase (LAH)activity. See, e.g., Jimenez (2001) Lipids 36:1169-1174, describing anoctaethylene glycol monododecyl ether-based mixed micellar assay fordetermining the lipid acyl hydrolase activity of a patatin. Pinsirodom(2000) J. Agric. Food Chem. 48:155-160, describes an exemplary lipidacyl hydrolase (LAH) patatin activity.

Turbidity assays to determine phospholipase activity are described,e.g., in Kauffmann (2001) “Conversion of Bacillus thermocatenulatuslipase into an efficient phospholipase with increased activity towardslong-chain fatty acyl substrates by directed evolution and rationaldesign,” Protein Engineering 14:919-928; Ibrahim (1995) “Evidenceimplicating phospholipase as a virulence factor of Candida albicans,”Infect. Immun. 63:1993-1998.

Methylumbelliferyl (fluorescent) phosphocholine assays to determinephospholipase activity are described, e.g., in Goode (1997) “Evidencefor cell surface and internal phospholipase activity in ascidian eggs,”Develop. Growth Differ. 39:655-660; Diaz (1999) “Directfluorescence-based lipase activity assay,” BioTechniques 27:696-700.

Amplex Red (fluorescent) Phospholipase Assays to determine phospholipaseactivity are available as kits, e.g., the detection ofphosphatidylcholine-specific phospholipase using an Amplex Redphosphatidylcholine-specific phospholipase assay kit from MolecularProbes Inc. (Eugene, Oreg.), according to manufacturer's instructions.Fluorescence is measured in a fluorescence microplate reader usingexcitation at 560±10 nm and fluorescence detection at 590±10 nm. Theassay is sensitive at very low enzyme concentrations.

Thin layer chromatography assays (TLC) to determine phospholipaseactivity are described, e.g., in Reynolds (1991) Methods in Enzymol.197:3-13; Taguchi (1975) “Phospholipase from Clostridium novyi typeA.I,” Biochim. Biophys. Acta 409:75-85. Thin layer chromatography (TLC)is a widely used technique for detection of phospholipase activity.Various modifications of this method have been used to extract thephospholipids from the aqueous assay mixtures. In some PLC assays thehydrolysis is stopped by addition of chloroform/methanol (2:1) to thereaction mixture. The unreacted starting material and the diacylglycerolare extracted into the organic phase and may be fractionated by TLC,while the head group product remains in the aqueous phase. For moreprecise measurement of the phospholipid digestion, radiolabeledsubstrates can be used (see, e.g., Reynolds (1991) Methods in Enzymol.197:3-13). The ratios of products and reactants can be used to calculatethe actual number of moles of substrate hydrolyzed per unit time. If allthe components are extracted equally, any losses in the extraction willaffect all components equally. Separation of phospholipid digestionproducts can be achieved by silica gel TLC withchloroform/methanol/water (65:25:4) used as a solvent system (see, e.g.,Taguchi (1975) Biochim. Biophys. Acta 409:75-85).p-Nitrophenylphosphorylcholine assays to determine phospholipaseactivity are described, e.g., in Korbsrisate (1999) J. Clin. Microbiol.37:3742-3745; Berka (1981) Infect. Immun. 34:1071-1074. This assay isbased on enzymatic hydrolysis of the substrate analogp-nitrophenylphosphorylcholine to liberate a yellow chromogenic compoundp-nitrophenol, detectable at 405 nm. This substrate is convenient forhigh-throughput screening.

A cytolytic assay can detect phospholipases with cytolytic activitybased on lysis of erythrocytes. Toxic phospholipases can interact witheukaryotic cell membranes and hydrolyze phosphatidylcholine andsphingomyelin, leading to cell lysis. See, e.g., Titball (1993)Microbiol. Rev. 57:347-366.

Hybrid (Chimeric) Phospholipases and Peptide Libraries

In one aspect, the invention provides hybrid phospholipases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asphospholipase substrates, receptors, enzymes. The peptide libraries ofthe invention can be used to identify formal binding partners oftargets, such as ligands, e.g., cytokines, hormones and the like. In oneaspect, the invention provides chimeric proteins comprising a signalsequence (SP) and/or catalytic domain (CD) of the invention and aheterologous sequence (see above).

The invention also provides methods for generating “improved” and hybridphospholipases using the nucleic acids and polypeptides of theinvention. For example, the invention provides methods for generatingenzymes that have activity, e.g., phospholipase activity (such as, e.g.,phospholipase A, B, C or D activity, patatin esterase activity, cleavageof a glycerolphosphate ester linkage, cleavage of an ester linkage in aphospholipid in a vegetable oil) at extreme alkaline pHs and/or acidicpHs, high and low temperatures, osmotic conditions and the like. Theinvention provides methods for generating hybrid enzymes (e.g., hybridphospholipases).

In one aspect, the methods of the invention produce new hybridpolypeptides by utilizing cellular processes that integrate the sequenceof a first polynucleotide such that resulting hybrid polynucleotidesencode polypeptides demonstrating activities derived from the firstbiologically active polypeptides. For example, the first polynucleotidescan be an exemplary nucleic acid sequence (e.g., SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, etc.) encoding an exemplaryphospholipase of the invention (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO: 8, etc.). The first nucleic acid can encode an enzymefrom one organism that functions effectively under a particularenvironmental condition, e.g. high salinity. It can be “integrated” withan enzyme encoded by a second polynucleotide from a different organismthat functions effectively under a different environmental condition,such as extremely high temperatures. For example, when the two nucleicacids can produce a hybrid molecule by e.g., recombination and/orreductive reassortment. A hybrid polynucleotide containing sequencesfrom the first and second original polynucleotides may encode an enzymethat exhibits characteristics of both enzymes encoded by the originalpolynucleotides. Thus, the enzyme encoded by the hybrid polynucleotidemay function effectively under environmental conditions shared by eachof the enzymes encoded by the first and second polynucleotides, e.g.,high salinity and extreme temperatures.

Alternatively, a hybrid polypeptide resulting from this method of theinvention may exhibit specialized enzyme activity not displayed in theoriginal enzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding phospholipase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized activities obtained from each of the originalenzymes, i.e. the type of bond on which the phospholipase acts and thetemperature at which the phospholipase functions. Thus, for example, thephospholipase may be screened to ascertain those chemicalfunctionalities which distinguish the hybrid phospholipase from theoriginal phospholipases, such as: (a) amide (peptide bonds), i.e.,phospholipases; (b) ester bonds, i.e., phospholipases and lipases; (c)acetals, i.e., glycosidases and, for example, the temperature, pH orsalt concentration at which the hybrid polypeptide functions.

Sources of the polynucleotides to be “integrated” with nucleic acids ofthe invention may be isolated from individual organisms (“isolates”),collections of organisms that have been grown in defined media(“enrichment cultures”), or, uncultivated organisms (“environmentalsamples”). The use of a culture-independent approach to derivepolynucleotides encoding novel bioactivities from environmental samplesis most preferable since it allows one to access untapped resources ofbiodiversity. “Environmental libraries” are generated from environmentalsamples and represent the collective genomes of naturally occurringorganisms archived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample that may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions that promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which hybrid polynucleotides may be preparedinclude prokaryotic microorganisms, such as Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. Polynucleotides may be isolated from environmentalsamples. Nucleic acid may be recovered without culturing of an organismor recovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halopliles, barophiles and acidophiles. Inone aspect, polynucleotides encoding phospholipase enzymes isolated fromextremophilic microorganisms are used to make hybrid enzymes. Suchenzymes may function at temperatures above 100° C. in, e.g., terrestrialhot springs and deep sea thermal vents, at temperatures below 0° C. in,e.g., arctic waters, in the saturated salt environment of, e.g., theDead Sea, at pH values around 0 in, e.g., coal deposits and geothermalsulfur-rich springs, or at pH values greater than 11 in, e.g., sewagesludge. For example, phospholipases cloned and expressed fromextremophilic organisms can show high activity throughout a wide rangeof temperatures and pHs.

Polynucleotides selected and isolated as described herein, including atleast one nucleic acid of the invention, are introduced into a suitablehost cell. A suitable host cell is any cell that is capable of promotingrecombination and/or reductive reassortment. The selectedpolynucleotides can be in a vector that includes appropriate controlsequences. The host cell can be a higher eukaryotic cell, such as amammalian cell, or a lower eulcaryotic cell, such as a yeast cell, orpreferably, the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,or electroporation (Davis et al., 1986).

Exemplary appropriate hosts include bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells suchas CHO, COS or Bowes melanoma; adenoviruses; and plant cells (see also,discussion above). The selection of an appropriate host forrecombination and/or reductive reassortment or just for expression ofrecombinant protein is deemed to be within the scope of those skilled inthe art from the teachings herein. Mammalian cell culture systems thatcan be employed for recombination and/or reductive reassortment or justfor expression of recombinant protein include, e.g., the COS-7 lines ofmonkey kidney fibroblasts, described in “SV40-transformed simian cellssupport the replication of early SV40 mutants” (Gluzman, 1981), theC127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectorscan comprise an origin of replication, a suitable promoter and enhancer,and necessary ribosome binding sites, polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

Host cells containing the polynucleotides of interest (for recombinationand/or reductive reassortment or just for expression of recombinantprotein) can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan. Theclones which are identified as having the specified enzyme activity maythen be sequenced to identify the polynucleotide sequence encoding anenzyme having the enhanced activity.

In another aspect, the nucleic acids and methods of the presentinvention can be used to generate novel polynucleotides for biochemicalpathways, e.g., pathways from one or more operons or gene clusters orportions thereof. For example, bacteria and many eukaryotes have acoordinated mechanism for regulating genes whose products are involvedin related processes. The genes are clustered, in structures referred toas “gene clusters,” on a single chromosome and are transcribed togetherunder the control of a single regulatory sequence, including a singlepromoter which initiates transcription of the entire cluster. Thus, agene cluster is a group of adjacent genes that are either identical orrelated, usually as to their function.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples.“Fosmids,” cosmids or bacterial artificial chromosome (BAC) vectors canbe used as cloning vectors. These are derived from E. coli f-factorwhich is able to stably integrate large segments of genomic DNA. Whenintegrated with DNA from a mixed uncultured environmental sample, thismakes it possible to achieve large genomic fragments in the form of astable “environmental DNA library.” Cosmid vectors were originallydesigned to clone and propagate large segments of genomic DNA. Cloninginto cosmid vectors is described in detail in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989). Once ligated into an appropriate vector, two or morevectors containing different polyketide synthase gene clusters can beintroduced into a suitable host cell. Regions of partial sequencehomology shared by the gene clusters will promote processes which resultin sequence reorganization resulting in a hybrid gene cluster. The novelhybrid gene cluster can then be screened for enhanced activities notfound in the original gene clusters.

Thus, in one aspect, the invention relates to a method for producing abiologically active hybrid polypeptide using a nucleic acid of theinvention and screening the polypeptide for an activity (e.g., enhancedactivity) by:

(1) introducing at least a first polynucleotide (e.g., a nucleic acid ofthe invention) in operable linkage and a second polynucleotide inoperable linkage, said at least first polynucleotide and secondpolynucleotide sharing at least one region of partial sequence homology,into a suitable host cell;

(2) growing the host cell under conditions which promote sequencereorganization resulting in a hybrid polynucleotide in operable linkage;

(3) expressing a hybrid polypeptide encoded by the hybridpolynucleotide;

(4) screening the hybrid polypeptide under conditions which promoteidentification of the desired biological activity (e.g., enhancedphospholipase activity); and

(5) isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

In vivo reassortment can be focused on “inter-molecular” processescollectively referred to as “recombination.” In bacteria it is generallyviewed as a “RecA-dependent” phenomenon. The invention can rely onrecombination processes of a host cell to recombine and re-assortsequences, or the cells' ability to mediate reductive processes todecrease the complexity of quasi-repeated sequences in the cell bydeletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process. Thus, in one aspect of theinvention, using the nucleic acids of the invention novelpolynucleotides are generated by the process of reductive reassortment.The method involves the generation of constructs containing consecutivesequences (original encoding sequences), their insertion into anappropriate vector, and their subsequent introduction into anappropriate host cell. The reassortment of the individual molecularidentities occurs by combinatorial processes between the consecutivesequences in the construct possessing regions of homology, or betweenquasi-repeated units. The reassortment process recombines and/or reducesthe complexity and extent of the repeated sequences, and results in theproduction of novel molecular species.

Various treatments may be applied to enhance the rate of reassortment.These could include treatment with ultra-violet light, or DNA damagingchemicals, and/or the use of host cell lines displaying enhanced levelsof “genetic instability”. Thus the reassortment process may involvehomologous recombination or the natural property of quasi-repeatedsequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. “Quasi-repeats” are repeats that are not restricted totheir original unit structure. Quasi-repeated units can be presented asan array of sequences in a construct; consecutive units of similarsequences. Once ligated, the junctions between the consecutive sequencesbecome essentially invisible and the quasi-repetitive nature of theresulting construct is now continuous at the molecular level. Thedeletion process the cell performs to reduce the complexity of theresulting construct operates between the quasi-repeated sequences. Thequasi-repeated units provide a practically limitless repertoire oftemplates upon which slippage events can occur. The constructscontaining the quasi-repeats thus effectively provide sufficientmolecular elasticity that deletion (and potentially insertion) eventscan occur virtually anywhere within the quasi-repetitive units. When thequasi-repeated sequences are all ligated in the same orientation, forinstance head to tail or vice versa, the cell cannot distinguishindividual units. Consequently, the reductive process can occurthroughout the sequences. In contrast, when for example, the units arepresented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, in one aspect of theinvention, the sequences to be reasserted are in the same orientation.Random orientation of quasi-repeated sequences will result in the lossof reassortment efficiency, while consistent orientation of thesequences will offer the highest efficiency. However, while having fewerof the contiguous sequences in the same orientation decreases theefficiency, it may still provide sufficient elasticity for the effectiverecovery of novel molecules. Constructs can be made with thequasi-repeated sequences in the same orientation to allow higherefficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following: a) Primers that include apoly-A head and poly-T tail which when made single-stranded wouldprovide orientation can be utilized. This is accomplished by having thefirst few bases of the primers made from RNA and hence easily removedRNase H. b) Primers that include unique restriction cleavage sites canbe utilized. Multiple sites, a battery of unique sequences, and repeatedsynthesis and ligation steps would be required. c) The inner few basesof the primer could be thiolated and an exonuclease used to produceproperly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by: 1) The use of vectors only stablymaintained when the construct is reduced in complexity. 2) The physicalrecovery of shortened vectors by physical procedures. In this case, thecloning vector would be recovered using standard plasmid isolationprocedures and size fractionated on either an agarose gel, or columnwith a low molecular weight cut off utilizing standard procedures. 3)The recovery of vectors containing interrupted genes which can beselected when insert size decreases. 4) The use of direct selectiontechniques with an expression vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, this process is not limitedto such nearly identical repeats.

The following is an exemplary method of the invention. Encoding nucleicacid sequences (quasi-repeats) are derived from three (3) species,including a nucleic acid of the invention. Each sequence encodes aprotein with a distinct set of properties, including an enzyme of theinvention. Each of the sequences differs by a single or a few base pairsat a unique position in the sequence. The quasi-repeated sequences areseparately or collectively amplified and ligated into random assembliessuch that all possible permutations and combinations are available inthe population of ligated molecules. The number of quasi-repeat unitscan be controlled by the assembly conditions. The average number ofquasi-repeated units in a construct is defined as the repetitive index(RI). Once formed, the constructs may, or may not be size fractionatedon an agarose gel according to published protocols, inserted into acloning vector, and transfected into an appropriate host cell. The cellsare then propagated and “reductive reassortment” is effected. The rateof the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired. Whether the reduction in RI ismediated by deletion formation between repeated sequences by an“intra-molecular” mechanism, or mediated by recombination-like eventsthrough “inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations. In oneaspect, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure. The polypeptides, e.g.,phospholipases, that are identified from such libraries can be used forvarious purposes, e.g., the industrial processes described herein and/orcan be subjected to one or more additional cycles of shuffling and/orselection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred means for slowing or haltingPCR amplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forphospholipase activity, to screen compounds as potential modulators ofactivity (e.g., potentiation or inhibition of enzyme activity), forantibodies that bind to a polypeptide of the invention, for nucleicacids that hybridize to a nucleic acid of the invention, and the like.

Immobilized Enzyme Solid Supports

The phospholipase enzymes, fragments thereof and nucleic acids thatencode the enzymes and fragments can be affixed to a solid support. Thisis often economical and efficient in the use of the phospholipases inindustrial processes. For example, a consortium or cocktail ofphospholipase enzymes (or active fragments thereof), which are used in aspecific chemical reaction, can be attached to a solid support anddunked into a process vat. The enzymatic reaction can occur. Then, thesolid support can be taken out of the vat, along with the enzymesaffixed thereto, for repeated use. In one embodiment of the invention,an isolated nucleic acid of the invention is affixed to a solid support.In another embodiment of the invention, the solid support is selectedfrom the group of a gel, a resin, a polymer, a ceramic, a glass, amicroelectrode and any combination thereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include Sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,Al₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support.

Other exemplary solid supports used to practice the invention comprisediatomaceous earth products and silicates. Some examples includeCELITE®KENITE®, DIACTIV®, PRIMISIL®, DIAFIL® diatomites and MICRO-CEL®,CALFLO®, SILASORB™, and CELKATE® synthetic calcium and magnesiumsilicates. Another example of a solid support is a cell, such as a redblood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the artfor immobilizing enzymes or fragments thereof, or nucleic acids, onto asolid support. Some examples of such methods include, e.g.,electrostatic droplet generation, electrochemical means, via adsorption,via covalent binding, via cross-linking, via a chemical reaction orprocess, via encapsulation, via entrapment, via calcium alginate, or viapoly (2-hydroxyethyl methacrylate). Like methods are described inMethods in Enzymology, Immobilized Enzymes and Cells, Part C. 1987.Academic Press. Edited by S. P. Colowick and N. O. Kaplan. Volume 136;and Immobilization of Enzymes and Cells. 1997. Humana Press. Edited byG. F. Bickerstaff. Series: Methods in Biotechnology, Edited by J. M.Walker.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into afirst component into at least a portion of a capillary of a capillaryarray. Each capillary of the capillary array can comprise at least onewall defining a lumen for retaining the first component. An air bubblecan be introduced into the capillary behind the first component. Asecond component can be introduced into the capillary, wherein thesecond component is separated from the first component by the airbubble. A sample of interest can be introduced as a first liquid labeledwith a detectable particle into a capillary of a capillary array,wherein each capillary of the capillary array comprises at least onewall defining a lumen for retaining the first liquid and the detectableparticle, and wherein the at least one wall is coated with a bindingmaterial for binding the detectable particle to the at least one wall.The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.

The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a microtiter plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “BioChips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a phospholipase gene. One or more, or, all the transcripts of a cellcan be measured by hybridization of a sample comprising transcripts ofthe cell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. “Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins.

The present invention can be practiced with any known “array,” alsoreferred to as a “microarray” or “nucleic acid array” or “polypeptidearray” or “antibody array” or “biochip,” or variation thereof. Arraysare generically a plurality of “spots” or “target elements,” each targetelement comprising a defined amount of one or more biological molecules,e.g., oligonucleotides, immobilized onto a defined area of a substratesurface for specific binding to a sample molecule, e.g., mRNAtranscripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a phospholipase of the invention. These antibodiescan be used to isolate, identify or quantify the phospholipases of theinvention or related polypeptides. These antibodies can be used toinhibit the activity of an enzyme of the invention. These antibodies canbe used to isolated polypeptides related to those of the invention,e.g., related phospholipase enzymes.

The antibodies can be used in immunoprecipitation, staining (e.g.,FACS), immunoaffinity columns, and the like. If desired, nucleic acidsequences encoding for specific antigens can be generated byimmunization followed by isolation of polypeptide or nucleic acid,amplification or cloning and immobilization of polypeptide onto an arrayof the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides can be used to generate antibodies which bindspecifically to the polypeptides of the invention. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to an animal, forexample, a nonhuman. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies which maybind to the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides from other organisms andsamples. In such techniques, polypeptides from the organism arecontacted with the antibody and those polypeptides which specificallybind the antibody are detected. Any of the procedures described abovemay be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, polypeptides (e.g., a kithaving at least one phospholipase of the invention) and/or antibodies(e.g., a kit having at least one antibody of the invention. The kitsalso can contain instructional material teaching the methodologies andindustrial uses of the invention, as described herein.

Industrial and Medical Uses of the Enzymes of the Invention

The invention provides many industrial uses and medical applicationsusing polypeptides of the invention, e.g., a phospholipase and otherenzymes of the invention, e.g., phospholipases A, B, C and D, patatins,including converting a non-hydratable phospholipid to a hydratable form,oil degumming, processing of oils from plants, fish, algae and the like,to name just a few applications. In any of these alternative industrialuses and medical applications, an enzymes can be added in a specificorder, e.g., phospholipases with differing specificities are added in aspecific order, for example, an enzyme with PC- and PE-hydrolyzingactivity is added first (or two enzymes are added, one withPC-hydrolyzing activity and the other with PE-hydrolyzing activity),then an enzyme with PI-hydrolyzing activity (e.g., PLC activity) isadded, or any combination thereof.

Any or all of the methods of the invention can be used on a “processscale”, e.g., an oil processes or refining on a scale from about 15,000;25,000; 50,000; 75,000; or 100,000 lbs of refined oil/day up to about 1,2, 3, 4, 5 or 6 or more million lbs refined oil/day.

Methods of using phospholipase enzymes in industrial applications arewell known in the art. For example, the phospholipases and methods ofthe invention can be used for the processing of fats and oils asdescribed, e.g., in JP Patent Application Publication H6-306386,describing converting phospholipids present in the oils and fats intowater-soluble substances containing phosphoric acid groups.

Phospholipases of the invention can be used to process plant oils andphospholipids such as those derived from or isolated from rice bran,soy, canola, palm, cottonseed, corn, palm kernel, coconut, peanut,sesame, sunflower. Phospholipases of the invention can be used toprocess essential oils, e.g., those from fruit seed oils, e.g.,grapeseed, apricot, borage, etc. Phospholipases of the invention can beused to process oils and phospholipids in different forms, includingcrude forms, degummed, gums, wash water, clay, silica, soapstock, andthe like. The phospholipids of the invention can be used to process highphosphorus oils, fish oils, animal oils, plant oils, algae oils and thelike. In any aspect of the invention, any time a phospholipase C can beused, an alternative comprises use of a phospholipase D of the inventionand a phosphatase (e.g., using a PLD/phosphatase combination to improveyield in a high phosphorus oil, such as a soy bean oil).

Phospholipases of the invention can be used to process and make edibleoils, biodiesel oils, liposomes for pharmaceuticals and cosmetics,structured phospholipids and structured lipids. Phospholipases of theinvention can be used in oil extraction. Phospholipases of the inventioncan be used to process and make various soaps.

Processing Edible Oils: Generation of 1,3-diacylglycerol (1,3 DAG)

The invention provides processes using enzyme(s) of the invention tomake 1,3-diacylglycerol (1,3 DAG). In one aspect, a phospholipase C orphospholipase D plus a phosphatase generates 1,2-diacylglycerol; thisimproves oil yield during edible oil refining. When used in a processthat includes a caustic neutralization step, for example as a causticrefining aid, as much as 70% of the 1,2-diacylglyceride (1,2-DAG)undergoes acyl migration and is converted to 1,3-DAG. 1,3-DAG possessesincreased health benefits and therefore the use of PLC as a causticrefining aid produces an oil with increased nutritional value.

The invention provides processes using enzyme(s) of the invention tomake and process edible oils, including generation of edible oils withincreased amounts of 1,3-DAG. Diacylglycerols are naturally occurringcompounds found in many edible oils. In one aspect of a method of theinvention, e.g., the oil degumming process, a base (caustic) causes theisomerization of 1,2-DAG, produced by PLC, into 1,3-DAG which provides anutritional health benefit over 1,2-DAG, e.g., the 1,3-DAG is burned asenergy instead of being stored as fat (as is 1,2-DAG). By adding the PLCat the front end of caustic refining process (and the acid and causticsubsequently), the methods of the invention generate an elevated levelof 1,3-DAG (decreasing 1,2-DAG). Nutritionally, 1,3-DAG is better foryou than 1,2-DAG. In alternative aspects, the invention comprises an oildegumming process using a PLC of the invention, whereby the finaldegummed oil product contains not less than 0.5%, 1.0%, 2.0% or 3.0% ormore 1,3-DAG.

Thus, the invention provides a process for making (throughinteresterification) a refined oil (e.g., a diacylglycerol oil),including edible oils, containing increased levels of 1,3-diacylglycerol(1,3-DAG), e.g., as illustrated in Example 13, where a phospholipase,such as an enzyme of the invention, is “front-loaded” or added beforeaddition of acid or caustic. The generation by enzymatic hydrolysis of aDAG from a triglyceride generates by interesterification 1,3 DAG from1,2 DAG. The 1,3 DAG-comprising edible oil shows different metaboliceffects compared to conventional edible oils. Differences in metabolicpathways between 1,3 DAG and either 1,2 DAG or triglycerides allow agreater portion of fatty acids from 1,3 diacylglycerol to be burned asenergy rather than being stored as fat. Clinical studies have shown thatregular consumption of DAG oil as part of a sensible diet can helpindividuals to manage their body weight and body fat. In addition,metabolism of 1,3 DAG reduces circulating postmeal triglycerides in thebloodstream. Since obesity and elevated blood lipids are associated asrisk factors for chronic diseases including cardiovascular disease andType II diabetes, these lifestyle-related health conditions may beimpacted in a beneficial manner with regular consumption of DAG oils.

Consumption of DAG-comprising oil can take place through a variety ofmeans. Thus, in one aspect, the invention provides a process using anenzyme of the invention for making a food, e.g., a baked good, havingincreased levels of 1,3-DAG diacylglycerol and baked goods comprisingdiacylglycerol oils. In one aspect, the baked goods are cookies, cakesand similar baked goods.

In alternative embodiments, combination of enzymes that can be used inthe methods of the invention, including the processing of edible oils,include (where one, several or all of the enzymes in the combinationcomprise an enzyme of the instant invention):

-   -   PLC+PI-PLC+PLA (PLA added after completion of PLC reactions);    -   PLD+phosphatase+PI-PLC followed by PLA; or,    -   PLC or (PLC+PI-PLC)+PLA specific for phosphatidic acid (all        enzymes added together or sequentially).

Oil Degumming and Vegetable Oil Processing

The enzymes of the invention (e.g., polypeptides of the invention havinglipase, phospholipase, esterase and/or glycosidase or equivalentactivity) can be used in various vegetable oil processing steps, such asin vegetable oil extraction, particularly, in the removal of“phospholipid gums” in a process called “oil degumming”.

These processes of the invention can be used on a “process scale”, e.g.,on a scale from about 15,000; 25,000; 50,000; 75,000; or 100,000 lbs ofrefined oil/day up to about 1, 2, 3, 4, 5 or 6 or more million lbsrefined oil/day.

In one aspect, the invention provides oil degumming processes comprisinguse of a phospholipase of the invention, e.g., a PLC of the invention.In one aspect, the process further comprises addition of anotherphospholipase (which can also be a phospholipase of the invention),e.g., another PLC, a PLA, a PLB, a PLB or a patatin of the invention, oran enzyme (which can also be an enzyme of the invention) having alysophospholipase-transacylase (LPTA) activity or lysophospholipase(LPL) activity and lysophospholipase-transacylase (LPTA), or acombination thereof, and/or a patatin-like phospholipase (which can alsobe an enzyme of the invention). In one aspect, all enzymes are addedtogether, or, alternatively, the enzymes are added in a specific order,e.g., PLC addition is followed by PLA and/or patatin addition; or, anenzyme or enzymes of the invention having PC and PE activity addedfirst, then PI PLC added second.

In one aspect, this process provides a yield improvement as a result ofthe phospholipase (e.g., PLC of the invention) treatment. In one aspect,this process provides an additional decrease of the phosphorus contentof the oil as a result of the phospholipase (e.g., PLA of the invention)treatment.

In one aspect, the invention provides processes comprising use of aphospholipase of the invention, e.g., a PLC of the invention, to reducegum mass and increase neutral oil (triglyceride) gain through reducedoil entrapment. In one aspect, the invention provides processescomprising use of a phospholipase of the invention, e.g., a PLC of theinvention, for increasing neutral oils and diacylglycerol (DAG)production to contribute to the oil phase. In alternative aspects,processes of the invention (e.g., degumming processes) may comprise oneor more other enzymes such as a protease, an amylase, a lipase, acutinase, another phospholipase (including, e.g., an enzyme of theinvention), a carbohydrase, a cellulase, a pectinase, a mannanase, anarabinase, a galactanase, a xylanase, an oxidase, e.g., a lactase,and/or a peroxidase, or polypeptides with equivalent activity, or acombination thereof.

The phospholipases of the invention can be used in various vegetable oilprocessing steps, such as in vegetable oil extraction, particularly, inthe removal of “phospholipid gums” in a process called “oil degumming,”as described above. The invention provides methods for processingvegetable oils from various sources, such as rice bran, soybeans,rapeseed, peanuts and other nuts, sesame, sunflower, palm and corn. Themethods can used in conjunction with processes based on extraction withas hexane, with subsequent refining of the crude extracts to edibleoils, including use of the methods and enzymes of the invention. Thefirst step in the refining sequence is the so-called “degumming”process, which serves to separate phosphatides by the addition of water.The material precipitated by degumming is separated and furtherprocessed to mixtures of lecithins. The commercial lecithins, such assoybean lecithin and sunflower lecithin, are semi-solid or very viscousmaterials. They consist of a mixture of polar lipids, mainlyphospholipids, and oil, mainly triglycerides.

The phospholipases of the invention can be used in any “degumming”procedure, including water degumming, ALCON oil degumming (e.g., forsoybeans), safinco degumming, “super degumming,” UF degumming, TOPdegumming, uni-degumming, dry degumming and ENZYMAX™ degumming. See,e.g., U.S. Pat. Nos. 6,355,693; 6,162,623; 6,103,505; 6,001,640;5,558,781; 5,264,367. Various “degumming” procedures incorporated by themethods of the invention are described in Bockisch, M. (1998) In Fatsand Oils Handbook, The extraction of Vegetable Oils (Chapter 5),345-445, AOCS Press, Champaign, Ill. The phospholipases of the inventioncan be used in the industrial application of enzymatic degumming oftriglyceride oils as described, e.g., in EP 513 709.

In one aspect, phospholipases of the invention are used to treatvegetable oils, e.g., crude oils, such as rice bran, soy, canola, flowerand the like. In one aspect, this improves the efficiency of thedegumming process. In one aspect, the invention provides methods forenzymatic degumming under conditions of low water, e.g., in the range ofbetween about 0.1% to 20% water, or, 0.5% to 10% water. In one aspect,this results in the improved separation of a heavy phase from the oilphase during centrifugation. The improved separation of these phases canresult in more efficient removal of phospholipids from the oil,including both hydratable and nonhydratable oils. In one aspect, thiscan produce a gum fraction that contains less entrained neutral oil(triglycerides), thereby improving the overall yield of oil during thedegumming process.

In one aspect, phospholipases of the invention, e.g., a polypeptidehaving PLC activity, are used to treat oils (e.g., vegetable oils,including crude oils, such as rice bran, soy, canola, flower and thelike), e.g., in degumming processes, to reduce gum mass and increaseneutral oil gain through reduced oil entrapment. In one aspect,phospholipases of the invention e.g., a polypeptide having PLC activity,are used for diacylglycerol (DAG) production and to contribute to theoil phase.

The phospholipases of the invention can be used in the industrialapplication of enzymatic degumming as described, e.g., in CA 1102795,which describes a method of isolating polar lipids from cereal lipids bythe addition of at least 50% by weight of water. This method is amodified degumming in the sense that it utilizes the principle of addingwater to a crude oil mixture.

In one aspect, the invention provides enzymatic processes comprising useof phospholipases of the invention (e.g., a PLC) comprising hydrolysisof hydrated phospholipids in oil at a temperature of about 20° C. to 40°C., at an alkaline pH, e.g., a pH of about pH 8 to pH 10, using areaction time of about 3 to 10 minutes. This can result in less than 10ppm final oil phosphorus levels. The invention also provides enzymaticprocesses comprising use of phospholipases of the invention (e.g., aPLC) comprising hydrolysis of hydratable and non-hydratablephospholipids in oil at a temperature of about 50° C. to 60° C., at a pHslightly below neutral, e.g., of about pH 5 to pH 6.5, using a reactiontime of about 30 to 60 minutes. This can result in less than 10 ppmfinal oil phosphorus levels.

In one aspect, the invention provides enzymatic processes that utilize aphospholipase C enzyme to hydrolyze a glyceryl phosphoester bond andthereby enable the return of the diacylglyceride portion ofphospholipids back to the oil, e.g., a vegetable, fish or algae oil (a“phospholipase C (PLC) caustic refining aid”); and, reduce thephospholipid content in a degumming step to levels low enough for highphosphorus oils to be physically refined (a “phospholipase C (PLC)degumming aid”). The two approaches can generate different values andhave different target applications.

In various exemplary processes of the invention, a number of distinctsteps compose the degumming process preceding the core bleaching anddeodorization refining processes. These steps include heating, mixing,holding, separating and drying. Following the heating step, water andoften acid are added and mixed to allow the insoluble phospholipid “gum”to agglomerate into particles which may be separated. While waterseparates many of the phosphatides in degumming, portions of thephospholipids are non-hydratable phosphatides (NHPs) present as calciumor magnesium salts. Degumming processes address these NHPs by theaddition of acid. Following the hydration of phospholipids, the oil ismixed, held and separated by centrifugation. Finally, the oil is driedand stored, shipped or refined, as illustrated, e.g., in FIG. 6. Theresulting gums are either processed further for lecithin products oradded back into the meal.

In various exemplary processes of the invention phosphorus levels arereduced low enough for physical refining. The separation process canresult in potentially higher yield losses than caustic refining.Additionally, degumming processes may generate waste products that maynot be sold as commercial lecithin, see, e.g., FIG. 7 for an exemplarydegumming process for physically refined oils. Therefore, theseprocesses have not achieved a significant share of the market andcaustic refining processes continue to dominate the industry for ricebran, soy, canola and sunflower. Note however, that a phospholipase Cenzyme employed in a special degumming process would decrease gumformation and return the diglyceride portion of the phospholipid back tothe oil.

In one aspect, the invention provides methods using a PLC of theinvention in the gum fraction. In one aspect of this method, oil isadded to the crude oil to create an emulsion that results in themovement of the phosphatidylcholine, phosphatidyl-ethanolamine andphosphatidylinositol into the aqueous phase (water degumming). Followingcentrifugation, these phospholipids are major components of the aqueousgum fraction. The phospholipids in the gum fraction can be treated withphospholipase C or phospholipase D plus phosphatase (or othercombinations, noted below) to generate diacylglycerol (DAG) and aphosphate ester. At this point, the DAG can be extracted from the othergum components and treated with a lipase under conditions suitable forthe transesterification of the DAG to produce a desired triacylglycerol(structured lipid).

In another aspect, the majority of the 1,2-DAG can be converted to1,3-DAG by increasing the pH of the gum following the PLC reaction, forexample, by adding caustic. The 1,3-DAG can then be extracted from thegum and reacted with a lipase under the appropriate conditions totransesterify the 1,3-DAG at the sn2 position to create the desiredstructured triacylglycerol.

In alternative aspects, the fatty acids used in the transesterificationreaction could come from a variety of sources including the free fattyacids found in the crude oil.

In one aspect, the phospholipids from water degumming are used incombination with a PLC of the invention to create structured lipids. Thewater-degummed oil can be exposed to a PLC and/or PLD (either or bothcan be enzymes of the invention) plus phosphatase or one of thesecombinations followed by PLA (can be an enzyme of the invention) toreduce the phosphorus to levels suitable for caustic or physicalrefining.

In alternative embodiments, combination of enzymes that can be used inthe methods of the invention, including these degumming processes,include (where one, several or all of the enzymes in the combinationcomprise an enzyme of the instant invention):

-   -   PLC+PI-PLC+PLA (PLA added after completion of PLC reactions);    -   PLD+phosphatase+PI-PLC followed by PLA; or,    -   PLC or (PLC+PI-PLC)+PLA specific for phosphatidic acid (all        enzymes added together or sequentially).

Caustic Refining

The invention provides processes using phospholipases (including enzymesof the invention) in caustic refining, where the enzymes are used ascaustic refining aids. In alternative aspects, a PLC or PLD and/or aphosphatase are used in the processes as a drop-in, either before,during, or after a caustic neutralization refining process (eithercontinuous or batch refining). The amount of enzyme added may varyaccording to the process. The water level used in the process can below, e.g., about 0.5 to 5%. Alternatively, caustic is be added to theprocess multiple times. In addition, the process may be performed atdifferent temperatures (25° C. to 70° C.), with different acidsorcaustics, and at varying pH (4-12). Concentrated solutions of caustic,e.g., more concentrated than the industrial standard of 11%, to decreasemass of gum can be used. In alternative aspects, the concentratedsolution of caustic is between about 12% and 50% concentrated, e.g.,about 20%, 30%, 40%, 50%, or 60% or more concentrated.

In one aspect, a phospholipase C enzyme of the invention hydrolyzes aphosphatide at a glyceryl phosphoester bond to generate a diglycerideand water-soluble phosphate compound. The hydrolyzed phosphatide movesto the aqueous phase, leaving the diglyceride in the oil phase, asillustrated in FIG. 8. One objective of the PLC “Caustic Refining Aid”is to convert the phospholipid gums formed during neutralization into adiacylglyceride that will migrate back into the oil phase. In contrast,one objective of the “PLC Degumming Aid” is to reduce the phospholipidsin crude oil to a phosphorus equivalent of less than 10 parts permillion (ppm).

Acids that may be used in a caustic refining process include, but arenot limited to, phosphoric, citric, ascorbic, sulfuric, fumaric, maleic,hydrochloric and/or acetic acids. Acids are used to hydratenon-hydratable phospholipids. Caustics that may be used include, but arenot limited to, KOH- and NaOH. Caustics are used to neutralize freefatty acids. Alternatively, phospholipases, or more particularly a PLCor a PLD and a phosphatase, are used for purification of phytosterolsfrom the gum/soapstock.

An alternate embodiment of the invention to add the phospholipase beforecaustic refining is to express the phospholipase in a plant. In anotherembodiment, the phospholipase is added during crushing of the plant,seeds or other plant part. Alternatively, the phospholipase is addedfollowing crushing, but prior to refining (i.e. in holding vessels). Inaddition, phospholipase is added as a refining pre-treatment, eitherwith or without acid.

Another embodiment of the invention, already described, is to add thephospholipase during a caustic refining process. In this process, thelevels of acid and caustic are varied depending on the level ofphosphorus and the level of free fatty acids. In addition, broadtemperature and pH ranges are used in the process, dependent upon thetype of enzyme used.

In another embodiment of the invention, the phospholipase is added aftercaustic refining (FIG. 9). In one instance, the phospholipase is addedin an intense mixer or in a retention mixer, prior to separation.Alternatively, the phospholipase is added following the heat step. Inanother embodiment, the phospholipase is added in the centrifugationstep. In an additional embodiment, the phospholipase is added to thesoapstock. Alternatively, the phospholipase is added to the washwater.In another instance, the phospholipase is added during the bleachingand/or deodorizing steps.

In one aspect, a phospholipase, e.g., a phospholipase C, enzyme of theinvention will hydrolyze the phosphatide from both hydratable andnon-hydratable phospholipids in neutralized crude and degummed oilsbefore bleaching and deodorizing. Exemplary “caustic refining” processesof the invention are illustrated in FIG. 9 and FIG. 13. FIG. 9 includesexemplary times, temperature and pHs for static mixer (30 to 60 min, 50to 60° C., pH 5 to 6.5) and retention mixer (3 to 10 min, 20 to 40° C.).The target enzyme can be applied as a drop-in product in the existingcaustic neutralization process, as illustrated in FIG. 9. In thisaspect, the enzyme will not be required to withstand extreme pH levelsif it is added after the addition of caustic. As illustrated in FIG. 13(an enzyme “front loading” exemplary process), any phospholipase,including, e.g., a phospholipase of the invention, such as a PLC, PLB,PLA and/or PLC, can be used in a crude oil degumming process, asdescribed, e.g., in Bailey's Industrial Oil & Fat Products v.4 (ed. Y.H. Hui). FIG. 14 and FIG. 15 illustrate variations of methods of theinvention where two or three centrifugation steps, respectively, areused in the process, which can be used to process any oil, e.g., avegetable oil such as crude soy oil, as shown in the figure. Theexemplary method of FIG. 15 has a centrifugation step before causticrefining (in addition to a centrifugation step after caustic refiningand before the water wash, and, after the water wash), while theexemplary method of FIG. 14 does not have a centrifugation step beforecaustic refining. FIG. 16 illustrates another exemplary variation ofthis process using acid treatment and having a centrifugation stepbefore a degumming step; this exemplary process can be used to processany oil, e.g., a vegetable oil such as crude soy oil, as shown in thefigure.

In one aspect, a phospholipase of the invention enables phosphorus to beremoved to the low levels acceptable in physical refining. In oneaspect, a PLC of the invention will hydrolyze the phosphatide from bothhydratable and non-hydratable phospholipids in crude oils beforebleaching and deodorizing. The target enzyme can be applied as a drop-inproduct in an existing degumming operation, see, e.g., FIG. 10. Givensub-optimal mixing in commercial equipment, it is likely that acid willbe required to bring the non-hydratable phospholipids in contact withthe enzyme at the oil/water interface. Therefore, in one aspect, anacid-stable PLC of the invention is used.

In one aspect, a PLC Degumming Aid process of the invention caneliminate losses in one, or all three, areas noted in Table 2. Lossesassociated in a PLC process can be estimated to be about 0.8% versus5.2% on a mass basis due to removal of the phosphatide.

TABLE 2 Losses Addressed by PLC Products Caustic Degumming Refining AidAid 1) Oil lost in gum 2.1% X X formation & separation 2) Saponified oil3.1% X in caustic addition 3) Oil trapped in <1.0% X X clay inbleaching* Total Yield Loss ~5.2% ~2.1% ~5.2%

Additional potential benefits of this process of the invention includethe following:

-   -   Reduced adsorbents—less adsorbents required with lower (<5 ppm)        phosphorus    -   Lower chemical usage—less chemical and processing costs        associated with hydration of non-hydratable phospholipids    -   Lower waste generation—less water required to remove phosphorus        from oil

Oils processed (e.g., “degunmmed”) by the methods of the inventioninclude plant oilseeds, e.g., soybean oil, rapeseed oil, rice bran oiland sunflower oil. In one aspect, the “PLC Caustic Refining Aid” of theinvention can save 1.2% over existing caustic refining processes. Therefining aid application addresses soy oil that has been degummed forlecithin and these are also excluded from the value/load calculations.

Performance targets of the processes of the invention can vary accordingto the applications and more specifically to the point of enzymeaddition, see Table 3.

TABLE 3 Performance Targets by Application Caustic Degumming RefiningAid Aid Incoming Oil Phosphorus <200 ppm* 600-1,400 ppm Levels Final OilPhosphorus <10 ppm^(†) <10 ppm Levels Hydratable & Non- Yes Yeshydratable gums Residence Time 3-10 minutes 30 minutes^(‡) LiquidFormulation Yes Yes Target pH 8-10^(‡‡‡) 5.0-5.5^(‡‡) Target Temperature20-40° C. ~50-60° C. Water Content <5% 1-1.25% Enzyme Formulation PurityNo lipase/protease¹ No lipase/protease Other Key Requirements Removal ofFe Removal of Fe *Water degummed oil ^(†)Target levels achieved inupstream caustic neutralization step but must be maintained ^(‡)1-2hours existing ^(‡‡)Acid degumming will require an enzyme that is stablein much more acidic conditions: pH at 2.3 for citric acid at 5%. (~RoehmUSPN 6,001,640). ^(‡‡‡)The pH of neutralized oil is NOT neutral. Testingat POS indicates that the pH will be in the alkaline range from 6.5-10(Dec. 9, 2002). Typical pH range needs to be determined.

Other processes that can be used with a phospholipase of the invention,e.g., a phospholipase A₁ can convert non-hydratable native phospholipidsto a hydratable form. In one aspect, the enzyme is sensitive to heat.This may be desirable, since heating the oil can destroy the enzyme.However, the degumming reaction must be adjusted to pH 4-5 and 60° C. toaccommodate this enzyme. At 300 Units/kg oil saturation dosage, thisexemplary process is successful at taking previously water-degummed oilphosphorus content down to ≦10 ppm P. Advantages can be decreased H₂0content and resultant savings in usage, handling and waste. Table 4lists exemplary applications for industrial uses for enzymes of theinvention:

TABLE 4 Exemplary Application Caustic Refining Degumming Aid Aid Soy oilw/lecithin production X Chemical refined soy oil, Sunflower oil, X XCanola oil Low phosphatide oils (e.g. palm) X

In addition to these various “degumming” processes, the phospholipasesof the invention can be used in any vegetable oil processing step. Forexample, phospholipase enzymes of the invention can be used in place ofPLA, e.g., phospholipase A2, in any vegetable oil processing step. Oilsthat are “processed” or “degummed” in the methods of the inventioninclude soybean oils, rapeseed oils, corn oils, oil from palm kernels,canola oils, sunflower oils, sesame oils, peanut oils, rice bran oil andthe like. The main products from this process include triglycerides.

In one exemplary process, when the enzyme is added to and reacted with acrude oil, the amount of phospholipase employed is about 10-10,000units, or, alternatively, about, 100-2,000 units, per 1 kg of crude oil.The enzyme treatment is conducted for 5 min to 10 hours at a temperatureof 30° C. to 90° C., or, alternatively, about, 40° C. to 70° C. Theconditions may vary depending on the optimum temperature of the enzyme.The amount of water added to dissolve the enzyme is 5-1,000 wt. partsper 100 wt. parts of crude oil, or, alternatively, about, 10 to 200 wt.parts per 100 wt. parts of crude oil.

Upon completion of such enzyme treatment, the enzyme liquid is separatedwith an appropriate means such as a centrifugal separator and theprocessed oil is obtained. Phosphorus-containing compounds produced byenzyme decomposition of gummy substances in such a process arepractically all transferred into the aqueous phase and removed from theoil phase. Upon completion of the enzyme treatment, if necessary, theprocessed oil can be additionally washed with water or organic orinorganic acid such as, e.g., acetic acid, citric acid, phosphoric acid,succinic acid, and equivalent acids, or with salt solutions.

In one exemplary process for ultra-filtration degumming, the enzyme isbound to a filter or the enzyme is added to an oil prior to filtrationor the enzyme is used to periodically clean filters.

In one exemplary process for a phospholipase-mediated physical refiningaid, water and enzyme are added to crude oil (e.g., crude vegetableoil). In one aspect, a PLC or a PLD of the invention and a phosphataseare used in the process. In phospholipase-mediated physical refining,the water level can be low, i.e. 0.5-5% and the process time should beshort (less than 2 hours, or, less than 60 minutes, or, less than 30minutes, or, less than 15 minutes, or, less than 5 minutes). The processcan be run at different temperatures (25° C. to 70° C.), using differentacids and/or caustics, at different pHs (e.g., 3-10).

In alternate aspects, water degumming is performed first to collectlecithin by centrifugation and then PLC or PLC and PLA of the inventionis added to remove non-hydratable phospholipids (the process should beperformed under low water concentration). In another aspect, waterdegumming of crude oil to less than 10 ppm (edible oils) and subsequentphysical refining (less than 50 ppm for biodiesel) is performed. In oneaspect, an emulsifier is added and/or the crude oil is subjected to anintense mixer to promote mixing. Alternatively, an emulsion-breaker isadded and/or the crude oil is heated to promote separation of theaqueous phase. In another aspect, an acid is added to promote hydrationof non-hydratable phospholipids. Additionally, phospholipases can beused to mediate purification of phytosterols from the gum/soapstock.

In one aspect, the invention provides compositions and methods (whichcan comprise use of phospholipases of the invention) for oil degummingcomprising using varying amounts of acid and base without makingsoapstock. Using this aspect of the invention for oil degumming, acid(including phosphoric and/or citric) can be used to hydratenon-hydratable phospholipids in high phosphorus oils (including soybean,canola, and sunflower). Once the phospholipids are hydrated, the pH ofthe aqueous phase can be raised using caustic addition: the amount ofcaustic added can create a favorable pH for enzyme activity but will notresult in the formation of a significant soapstock fraction in the oil.Because a soapstock is not formed, the free fatty acids in the oil canbe removed downstream, following the degumming step, during bleachingand deodorization.

Enzymes of the invention are used to improve oil extraction and oildegumming (e.g., vegetable oils). In one aspect, a PLC of the inventionand at least one plant cell wall degrader (e.g., a cellulase, ahemicellulase or the like, to soften walls and increase yield atextraction) is used in a process of the invention. In this exemplaryapproach to using enzymes of the invention to improve oil extraction andoil degumming, a phospholipase C of the invention as well as otherhydrolases (e.g., a cellulase, a hemicellulase, an esterase, a proteaseand/or a phosphatase) are used during the crushing steps associated withoil production (including but not limited to soybean, canola, sunflower,rice bran oil). By using enzymes prior to or in place of solventextraction, it is possible to increase oil yield and reduce the amountof hydratable and non-hydratable phospholipids in the crude oil. Thereduction in non-hydratable phospholipids may result from conversion ofpotentially non-hydratable phospholipids to diacylglycerol andcorresponding phosphate-ester prior to complexation with calcium ormagnesium. The overall reduction of phospholipids in the crude oil willresult in improved yields during refining with the potential foreliminating the requirement for a separate degumming step prior tobleaching and deodorization.

In one aspect, the invention provides processes using a phospholipase ofthe invention (e.g., a phospholipase-specific phosphohydrolase of theinvention), or another phospholipase, in a modified “organic refiningprocess,” which can comprise addition of the enzyme (e.g., a PLC) in acitric acid holding tank.

The enzymes of the invention can be used in any oil processing method,e.g., degumming or equivalent processes. For example, the enzymes of theinvention can be used in processes as described in U.S. Pat. Nos.5,558,781; 5,264,367; 6,001,640. The process described in U.S. Pat. No.5,558,781 uses either phospholipase A1, A2 or B, essentially breakingdown lecithin in the oil that behaves as an emulsifier.

The enzymes and methods of the invention can be used in processes forthe reduction of phosphorus-containing components in edible oilscomprising a high amount of non-hydratable phosphorus by using of aphospholipase of the invention, e.g., a polypeptide having aphospholipase A and/or B activity, as described, e.g., in EP PatentNumber: EP 0869167. In one aspect, the edible oil is a crude oil, aso-called “non-degummed oil.” In one aspect, the method treat anon-degummed oil, including pressed oils or extracted oils, or a mixturethereof, from, e.g., rapeseed, soybean, sesame, peanut, corn, rice branor sunflower. The phosphatide content in a crude oil can vary from 0.5to 3% w/w corresponding to a phosphorus content in the range of 200 to1200 ppm, or, in the range of 250 to 1200 ppm. Apart from thephosphatides, the crude oil can also contains small concentrations ofcarbohydrates, sugar compounds and metal/phosphatide acid complexes ofCa, Mg and Fe. In one aspect, the process comprises treatment of aphospholipid or lysophospholipid with the phospholipase of the inventionso as to hydrolyze fatty acyl groups. In one aspect, the phospholipid orlysophospholipid comprises lecithin or lysolecithin. In one aspect ofthe process the edible oil has a phosphorus content from between about50 to 250 ppm, and the process comprises treating the oil with aphospholipase of the invention so as to hydrolyze a major part of thephospholipid and separating an aqueous phase containing the hydrolyzedphospholipid from the oil. In one aspect, prior to the enzymaticdegumming process the oil is water-degummed. In one aspect, the methodsprovide for the production of an animal feed comprising mixing thephospholipase of the invention with feed substances and at least onephospholipid.

The enzymes and methods of the invention can be used in processes of oildegumming as described, e.g., in WO 98/18912. The phospholipases of theinvention can be used to reduce the content of phospholipid in an edibleoil. The process can comprise treating the oil with a phospholipase ofthe invention to hydrolyze a major part of the phospholipid andseparating an aqueous phase containing the hydrolyzed phospholipid fromthe oil. This process is applicable to the purification of any edibleoil, which contains a phospholipid, e.g. vegetable oils, such as soybeanoil, rice bran oil, rapeseed oil and sunflower oil, fish oils, algae andanimal oils and the like. Prior to the enzymatic treatment, thevegetable oil is preferably pretreated to remove slime (mucilage), e.g.by wet refining. The oil can contain between about 50 to 250 ppm, orbetween 50 to about 1500 ppm, or more, of phosphorus, as phospholipid atthe start of the treatment with phospholipase, and the process of theinvention can reduce this value to below between about 5 to 10 ppm.

The enzymes of the invention can be used in processes as described in JPApplication No.: H5-132283, filed Apr. 25, 1993, which comprises aprocess for the purification of oils and fats comprising a step ofconverting phospholipids present in the oils and fats into water-solublesubstances containing phosphoric acid groups and removing them aswater-soluble substances. An enzyme action is used for the conversioninto water-soluble substances. An enzyme having a phospholipase Cactivity is preferably used as the enzyme.

The enzymes of the invention can be used in processes as described asthe “Organic Refining Process,” (ORP) (IPH, Omaha, Nebr.) which is amethod of refining seed oils. ORP may have advantages over traditionalchemical refining, including improved refined oil yield, value addedco-products, reduced capital costs and lower environmental costs.

The enzymes of the invention can be used in processes for the treatmentof an oil or fat, animal or vegetal, raw, semi-processed or refined,comprising adding to such oil or fat at least one enzyme of theinvention that allows hydrolyzing and/or depolymerizing thenon-glyceridic compounds contained in the oil, as described, e.g., in EPApplication number: 82870032.8. Exemplary methods of the invention forhydrolysis and/or depolymerization of non-glyceridic compounds in oilsare:

-   1) The addition and mixture in oils and fats of an enzyme of the    invention or enzyme complexes previously dissolved in a small    quantity of appropriate solvent (for example water). A certain    number of solvents are possible, but a non-toxic and suitable    solvent for the enzyme is chosen. This addition may be done in    processes with successive loads, as well as in continuous processes.    The quantity of enzyme(s) necessary to be added to oils and fats,    according to this process, may range, depending on the enzymes and    the products to be processed, from between about 5 to 400 ppm, or    between about 20 to 400 ppm; e.g., 0.005 kg to 0.4 kg of enzyme for    1000 kg of oil or fat, and preferably from 5 to 100 ppm, i.e., from    0.005 to 0.1 kg of enzyme for 1000 kg of oil, these values being    understood to be for concentrated enzymes, i.e., without diluent or    solvent.-   2) Passage of the oil or fat through a fixed or insoluble filtering    bed of enzyme(s) of the invention on solid or semi-solid supports,    preferably presenting a porous or fibrous structure. In this    technique, the enzymes are trapped in the micro-cavities of the    porous or fibrous structure of the supports. These consist, for    example, of resins or synthetic polymers, cellulose carbonates, gels    such as agarose, filaments of polymers or copolymers with porous    structure, trapping small droplets of enzyme in solution in their    cavities. Concerning the enzyme concentration, it is possible to go    up to the saturation of the supports.-   3) Dispersion of the oils and fats in the form of fine droplets, in    a diluted enzymatic solution, in alternative aspects containing    between about 0.05 to 4%, or containing between about 0.2 to 4%, in    volume of an enzyme of the invention. This technique is described,    e.g., in Belgian patent No. 595,219. A cylindrical column with a    height of several meters, with conical lid, is filled with a diluted    enzymatic solution. For this purpose, a solvent that is non-toxic    and non-miscible in the oil or fat to be processed, preferably    water, is chosen. The bottom of the column is equipped with a    distribution system in which the oil or fat is continuously injected    in an extremely divided form (approximately 10,000 flux per m²).    Thus an infinite number of droplets of oil or fat are formed, which    slowly rise in the solution of enzymes and meet at the surface, to    be evacuated continuously at the top of the conical lid of the    reactor.

Palm oil can be pre-treated before treatment with an enzyme of theinvention. For example, about 30 kg of raw palm oil is heated to +50° C.1% solutions were prepared in distilled water with cellulases andpectinases. 600 g of each of these was added to aqueous solutions of theoil under strong agitation for a few minutes. The oil is then kept at+50° C. under moderate agitation, for a total reaction time of twohours. Then, temperature is raised to +90° C. to deactivate the enzymesand prepare the mixture for filtration and further processing. The oilis dried under vacuum and filtered with a filtering aid.

The enzymes of the invention can be used in processes as described in EPpatent EP 0 513 709 B2. For example, the invention provides a processfor the reduction of the content process for the reduction of thecontent of phosphorus-containing components in animal and vegetable oilsby enzymatic decomposition using a phospholipase of the invention. Inalternative aspects, predemucilaginated animal and vegetable oil with aphosphorus content of between about of 50 to 1500 ppm, or, between about50 to 250 ppm, is agitated with an organic carboxylic acid and the pHvalue of the resulting mixture set to between about pH 4 to pH 6, anenzyme solution which contains phospholipase A₁, A₂, or B of theinvention is added to the mixture in a mixing vessel under turbulentstirring and with the formation of fine droplets, where an emulsion with0.5 to 5% by weight relative to the oil is formed, said emulsion beingconducted through at least one subsequent reaction vessel underturbulent motion during a reaction time of 0.1 to 10 hours attemperatures in the range of 20 to 80° C. and where the treated oil,after separation of the aqueous solution, has a phosphorus content under5 ppm.

The organic refining process is applicable to both crude and degummedoil. The process uses inline addition of an organic acid undercontrolled process conditions, in conjunction with conventionalcentrifugal separation. The water separated naturally from the vegetableoil phospholipids (“VOP”) is recycled and reused. The total water usagecan be substantially reduced as a result of the Organic RefiningProcess.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,162,623. In this exemplary methods, the invention provides anamphiphilic enzyme. It can be immobilized, e.g., by preparing anemulsion containing a continuous hydrophobic phase and a dispersedaqueous phase containing the enzyme and a carrier for the enzyme andremoving water from the dispersed phase until this phase turns intosolid enzyme coated particles. The enzyme can be a lipase. Theimmobilized lipase can be used for reactions catalyzed by lipase such asinteresterification of mono-, di- or triglycerides, de-acidification ofa triglyceride oil, or removal of phospholipids from a triglyceride oilwhen the lipase is a phospholipase. The aqueous phase may contain afermentation liquid, an edible triglyceride oil may be the hydrophobicphase, and carriers include sugars, starch, dextran, water solublecellulose derivatives and fermentation residues. This exemplary methodcan be used to process triglycerides, diglycerides, monoglycerides,glycerol, phospholipids, glycolipids or fatty acids, which may be in thehydrophobic phase. In one aspect, the process for the removal ofphospholipids from triglyceride oil comprising mixing a triglyceride oilcontaining phospholipids with a preparation containing a phospholipaseof the invention; hydrolyzing the phospholipids to lysophospholipid;separating the hydrolyzed phospholipids from the oil, wherein thephospholipase is an immobilized phospholipase.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,127,137. This exemplary method hydrolyzes both fatty acyl groups inintact phospholipid. The phospholipase of the invention used in thisexemplary method has no lipase activity and is active at very low pH.These properties make it very suitable for use in oil degumming, asenzymatic and alkaline hydrolysis (saponification) of the oil can bothbe suppressed. In one aspect, the invention provides a process forhydrolyzing fatty acyl groups in a phospholipid or lysophospholipidcomprising treating the phospholipid or lysophospholipid with thephospholipase that hydrolyzes both fatty acyl groups in a phospholipidand is essentially free of lipase activity. In one aspect, thephospholipase of the invention has a temperature optimum at about 50°C., measured at pH 3 to pH 4 for 10 minutes, and a pH optimum of aboutpH 3, measured at 40° C. for about 10 minutes. In one aspect, thephospholipid or lysophospholipid comprises lecithin or lysolecithin. Inone aspect, after hydrolyzing a major part of the phospholipid, anaqueous phase containing the hydrolyzed phospholipid is separated fromthe oil. In one aspect, the invention provides a process for removingphospholipid from an edible oil, comprising treating the oil at pH 1.5to 3 with a dispersion of an aqueous solution of the phospholipase ofthe invention, and separating an aqueous phase containing the hydrolyzedphospholipid from the oil. In one aspect, the oil is treated to removemucilage prior to the treatment with the phospholipase. In one aspect,the oil prior to the treatment with the phospholipase contains thephospholipid in an amount corresponding to 50 to 250 ppm of phosphorus.In one aspect, the treatment with phospholipase is done at 30° C. to 45°C. for 1 to 12 hours at a phospholipase dosage of 0.1 to 10 mg/l in thepresence of 0.5 to 5% of water.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,025,171. In this exemplary methods, enzymes of the invention areimmobilized by preparing an emulsion containing a continuous hydrophobicphase, such as a triglyceride oil, and a dispersed aqueous phasecontaining an amphiphilic enzyme, such as lipase or a phospholipase ofthe invention, and carrier material that is partly dissolved and partlyundissolved in the aqueous phase, and removing water from the aqueousphase until the phase turns into solid enzyme coated carrier particles.The undissolved part of the carrier material may be a material that isinsoluble in water and oil, or a water soluble material in undissolvedform because the aqueous phase is already saturated with the watersoluble material. The aqueous phase may be formed with a crude lipasefermentation liquid containing fermentation residues and biomass thatcan serve as carrier materials. Immobilized lipase is useful for esterre-arrangement and de-acidification in oils. After a reaction, theimmobilized enzyme can be regenerated for a subsequent reaction byadding water to obtain partial dissolution of the carrier, and with theresultant enzyme and carrier-containing aqueous phase dispersed in ahydrophobic phase evaporating water to again form enzyme coated carrierparticles.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,143,545. This exemplary method is used for reducing the content ofphosphorus containing components in an edible oil comprising a highamount of non-hydratable phosphorus content using a phospholipase of theinvention. In one aspect, the method is used to reduce the content ofphosphorus containing components in an edible oil having anon-hydratable phosphorus content of at least 50 ppm measured bypre-treating the edible oil, at 60° C., by addition of a solutioncomprising citric acid monohydrate in water (added water vs. oil equals4.8% w/w; (citric acid) in water phase=106 mM, in water/oil emulsion=4.6mM) for 30 minutes; transferring 10 ml of the pre-treated water in oilemulsion to a tube; heating the emulsion in a boiling water bath for 30minutes; centrifuging at 5000 rpm for 10 minutes, transferring about 8ml of the upper (oil) phase to a new tube and leaving it to settle for24 hours; and drawing 2 g from the upper clear phase for measurement ofthe non-hydratable phosphorus content (ppm) in the edible oil. Themethod also can comprise contacting an oil at a pH from about pH 5 to 8with an aqueous solution of a phospholipase A or B of the invention(e.g., PLA1, PLA2, or a PLB), which solution is emulsified in the oiluntil the phosphorus content of the oil is reduced to less than 11 ppm,and then separating the aqueous phase from the treated oil.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.5,532,163. The invention provides processes for the refining of oil andfat by which phospholipids in the oil and fat to be treated can bedecomposed and removed efficiently. In one aspect, the inventionprovides a process for the refining of oil and fat which comprisesreacting, in an emulsion, the oil and fat with an enzyme of theinvention, e.g., an enzyme having an activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids (e.g., a PLA2 ofthe invention); and another process in which the enzyme-treated oil andfat is washed with water or an acidic aqueous solution. In one aspect,the acidic aqueous solution to be used in the washing step is a solutionof at least one acid, e.g., citric acid, acetic acid, phosphoric acidand salts thereof. In one aspect, the emulsified condition is formedusing 30 weight parts or more of water per 100 weight parts of the oiland fat. Since oil and fat can be purified without employing theconventional alkali refining step, generation of washing waste water andindustrial waste can be reduced. In addition, the recovery yield of oilis improved because loss of neutral oil and fat due to their inclusionin these wastes does not occur in the inventive process. In one aspect,the invention provides a process for refining oil and fat containingabout 100 to 10,000 ppm of phospholipids which comprises: reacting, inan emulsified condition, said oil and fat with an enzyme of theinvention having activity to decompose glycerol-fatty acid ester bondsin glycerophospholipids. In one aspect, the invention provides processesfor refining oil and fat containing about 100 to 10,000 ppm ofphospholipids which comprises reacting, in an emulsified condition, oiland fat with an enzyme of the invention having activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids; andsubsequently washing the treated oil and fat with a washing water.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.5,264,367. The content of phosphorus-containing components and the ironcontent of an edible vegetable or animal oil, such as an oil, e.g.,soybean oil, which has been wet-refined to remove mucilage, are reducedby enzymatic decomposition by contacting the oil with an aqueoussolution of an enzyme of the invention, e.g., a phospholipase A1, A2, orB, and then separating the aqueous phase from the treated oil. In oneaspect, the invention provides an enzymatic method for decreasing thecontent of phosphorus- and iron-containing components in oils, whichhave been refined to remove mucilage. An oil, which has been refined toremove mucilage, can be treated with an enzyme of the invention, e.g.,phospholipase C, A1, A2, or B. Phosphorus contents below 5 ppm and ironcontents below 1 ppm can be achieved. The low iron content can beadvantageous for the stability of the oil.

The phospholipases and methods of the invention can also be used forpreparing transesterified oils, as described, e.g., in U.S. Pat. No.5,288,619. The invention provides methods for enzymatictransesterification for preparing a margarine oil having both lowtrans-acid and low intermediate chain fatty acid content. The methodincludes the steps of providing a transesterification reaction mixturecontaining a stearic acid source material and an edible liquid vegetableoil, transesterifying the stearic acid source material and the vegetableoil using a 1-, 3-positionally specific lipase, and then finallyhydrogenating the fatty acid mixture to provide a recycle stearic acidsource material for a recyclic reaction with the vegetable oil. Theinvention also provides a counter-current method for preparing atransesterified oil. The method includes the steps of providing atransesterification reaction zone containing a 1-, 3-positionallyspecific lipase, introducing a vegetable oil into thetransesterification zone, introducing a stearic acid source material,conducting a supercritical gas or subcritical liquefied gascounter-current fluid, carrying out a transesterification reaction ofthe triglyceride stream with the stearic acid or stearic acid monoesterstream in the reaction zone, withdrawing a transesterified triglyceridemargarine oil stream, withdrawing a counter-current fluid phase,hydrogenating the transesterified stearic acid or stearic acid monoesterto provide a hydrogenated recycle stearic acid source material, andintroducing the hydrogenated recycle stearic acid source material intothe reaction zone.

In one aspect, the highly unsaturated phospholipid compound may beconverted into a triglyceride by appropriate use of a phospholipase C ofthe invention to remove the phosphate group in the sn-3 position,followed by 1,3 lipase acyl ester synthesis. The 2-substitutedphospholipid may be used as a functional food ingredient directly, ormay be subsequently selectively hydrolyzed in reactor 160 using animmobilized phospholipase C of the invention to produce a 1-diglyceride,followed by enzymatic esterification as described herein to produce atriglyceride product having a 2-substituted polyunsaturated fatty acidcomponent.

The phospholipases and methods of the invention can also be used in avegetable oil enzymatic degumming process as described, e.g., in U.S.Pat. No. 6,001,640. This method of the invention comprises a degummingstep in the production of edible oils. Vegetable oils from whichhydratable phosphatides have been eliminated by a previous aqueousdegumming process are freed from non-hydratable phosphatides byenzymatic treatment using a phospholipase of the invention. The processcan be gentle, economical and environment-friendly. Phospholipases thatonly hydrolyze lysolecithin, but not lecithin, are used in thisdegumming process.

In one aspect, to allow the enzyme of the invention to act, both phases,the oil phase and the aqueous phase that contain the enzyme, must beintimately mixed. It may not be sufficient to merely stir them. Gooddispersion of the enzyme in the oil is aided if it is dissolved in asmall amount of water, e.g., 0.5-5 weight-% (relative to the oil), andemulsified in the oil in this form, to form droplets of less than 10micrometers in diameter (weight average). The droplets can be smallerthan 1 micrometer. Turbulent stirring can be done with radial velocitiesabove 100 cm/sec. The oil also can be circulated in the reactor using anexternal rotary pump. The aqueous phase containing the enzyme can alsobe finely dispersed by means of ultrasound action. A dispersionapparatus can be used.

The enzymatic reaction probably takes place at the border surfacebetween the oil phase and the aqueous phase. It is the goal of all thesemeasures for mixing to create the greatest possible surface for theaqueous phase which contains the enzyme. The addition of surfactantsincreases the microdispersion of the aqueous phase. In some cases,therefore, surfactants with HLB values above 9, such as Na-dodecylsulfate, are added to the enzyme solution, as described, e.g., in EP-A 0513 709. A similar effective method for improving emulsification is theaddition of lysolecithin. The amounts added can lie in the range of0.001% to 1%, with reference to the oil. The temperature during enzymetreatment is not critical. Temperatures between 20° C. and 80° C. can beused, but the latter can only be applied for a short time. In thisaspect, a phospholipase of the invention having a good temperatureand/or low pH tolerance is used. Application temperatures of between 30°C. and 50° C. are optimal. The treatment period depends on thetemperature and can be kept shorter with an increasing temperature.Times of 0.1 to 10 hours, or, 1 to 5 hours are generally sufficient. Thereaction takes place in a degumming reactor, which can be divided intostages, as described, e.g., in DE-A 43 39 556. Therefore continuousoperation is possible, along with batch operation. The reaction can becarried out in different temperature stages. For example, incubation cantake place for 3 hours at 40° C., then for 1 hour at 60° C. If thereaction proceeds in stages, this also opens up the possibility ofadjusting different pH values in the individual stages. For example, inthe first stage the pH of the solution can be adjusted to 7, forexample, and in a second stage to 2.5, by adding citric acid. In atleast one stage, however, the pH of the enzyme solution must be below 4,or, below 3. If the pH was subsequently adjusted below this level, adeterioration of effect may be found. Therefore the citric acid can beadded to the enzyme solution before the latter is mixed into the oil.

After completion of the enzyme treatment, the enzyme solution, togetherwith the decomposition products of the NHP contained in it, can beseparated from the oil phase, in batches or continuously, e.g., by meansof centrifugation. Since the enzymes are characterized by a high levelof stability and the amount of the decomposition products contained inthe solution is slight (they may precipitate as sludge) the same aqueousenzyme phase can be used several times. There is also the possibility offreeing the enzyme of the sludge, see, e.g., DE-A 43 39 556, so that anenzyme solution which is essentially free of sludge can be used again.In one aspect of this degumming process, oils which contain less than 15ppm phosphorus are obtained. One goal is phosphorus contents of lessthan 10 ppm; or, less than 5 ppm. With phosphorus contents below 10 ppm,further processing of the oil according to the process of distillativede-acidification is easily possible. A number of other ions, such asmagnesium, calcium, zinc, as well as iron, can be removed from the oil,e.g., below 0.1 ppm. Thus, this product possesses ideal prerequisitesfor good oxidation resistance during further processing and storage.

The phospholipases and methods of the invention also can also be usedfor reducing the amount of phosphorus-containing components in vegetableand animal oils as described, e.g., in EP patent EP 0513709. In thismethod, the content of phosphorus-containing components, especiallyphosphatides, such as lecithin, and the iron content in vegetable andanimal oils, which have previously been deslimed, e.g. soya oil, arereduced by enzymatic breakdown using a phospholipase A1, A2 or B of theinvention.

The phospholipases and methods of the invention can also be used forrefining fat or oils as described, e.g., in JP 06306386. The inventionprovides processes for refining a fat or oil comprising a step ofconverting a phospholipid in a fat or an oil into a water-solublephosphoric-group-containing substance and removing this substance. Theaction of an enzyme of the invention (e.g., a PLC) is utilized toconvert the phospholipid into the substance. Thus, it is possible torefine a fat or oil without carrying out an alkali refining step fromwhich industrial wastes containing alkaline waste water and a largeamount of oil are produced. Improvement of yields can be accomplishedbecause the loss of neutral fat or oil from escape with the wastes canbe reduced to zero. In one aspect, gummy substances are converted intowater-soluble substances and removed as water-soluble substances byadding an enzyme of the invention having a phospholipase C activity inthe stage of degumming the crude oil and conducting enzymatic treatment.In one aspect, the phospholipase C of the invention has an activity thatcuts ester bonds of glycerin and phosphoric acid in phospholipids. Ifnecessary, the method can comprise washing the enzyme-treated oil withwater or an acidic aqueous solution. In one aspect, the enzyme of theinvention is added to and reacted with the crude oil. The amount ofphospholipase C employed can be 10 to 10,000 units, or, about 100 to2,000 units, per 1 kg of crude oil.

The phospholipases and methods of the invention can also be used forwater-degumming processes as described, e.g., in Dijkstra, Albert J., etal., Oleagineux, Corps Gras, Lipides (1998), 5(5), 367-370. In thisexemplary method, the water-degumming process is used for the productionof lecithin and for dry degumming processes using a degumming acid andbleaching earth. This method may be economically feasible only for oilswith a low phosphatide content, e.g., palm oil, lauric oils, etc. Forseed oils having a high NHP-content, the acid refining process is used,whereby this process is carried out at the oil mill to allow gumdisposal via the meal. In one aspect, this acid refined oil is apossible “polishing” operation to be carried out prior to physicalrefining.

The phospholipases and methods of the invention can also be used fordegumming processes as described, e.g., in Dijkstra, et al., Res. Dev.Dep., N.V. Vandemoortele Coord. Cent., Izegem, Belg. JAOCS, J. Am. OilChem. Soc. (1989), 66:1002-1009. In this exemplary method, the totaldegumming process involves dispersing an acid such as H₃PO₄ or citricacid into soybean oil, allowing a contact time, and then mixing a basesuch as caustic soda or Na silicate into the acid-in-oil emulsion. Thiskeeps the degree of neutralization low enough to avoid forming soaps,because that would lead to increased oil loss. Subsequently, the oilpassed to a centrifugal separator where most of the gums are removedfrom the oil stream to yield a gum phase with minimal oil content. Theoil stream is then passed to a second centrifugal separator to removeall remaining gums to yield a dilute gum phase, which is recycled.Washing and drying or in-line alkali refining complete the process.After the adoption of the total degumming process, in comparison withthe classical alkali refining process, an overall yield improvement ofabout 0.5% is realized. The totally degummed oil can be subsequentlyalkali refined, bleached and deodorized, or bleached and physicallyrefined.

The phospholipases and methods of the invention can also be used for theremoval of nonhydratable phospholipids from a plant oil, e.g., soybeanoil, as described, e.g., in Hvolby, et al., Sojakagefabr., Copenhagen,Den., J. Amer. Oil Chem. Soc. (1971) 48:503-509. In this exemplarymethod, water-degummed oil is mixed at different fixed pH values withbuffer solutions with and without Ca⁺⁺, Mg/Ca-binding reagents, andsurfactants. The nonhydratable phospholipids can be removed in anonconverted state as a component of micelles or of mixed emulsifiers.Furthermore, the nonhydratable phospholipids are removable by conversioninto dissociated forms, e.g., by removal of Mg and Ca from thephosphatidates, which can be accomplished by acidulation or by treatmentwith Mg/Ca-complexing or Mg/Ca-precipitating reagents. Removal orchemical conversion of the nonhydratable phospholipids can result inreduced emulsion formation and in improved separation of the deacidifiedoil from the emulsion layer and the soapstock.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., Buchold, et al.,Frankfurt/Main, Germany. Fett Wissenschaft Technologie (1993), 95(8),300-304. In this exemplary process of the invention for the degumming ofedible vegetable oils, aqueous suspensions of an enzyme of theinvention, e.g., phospholipase A2, is used to hydrolyze the fatty acidbound at the sn2 position of the phospholipid, resulting in1-acyl-lysophospholipids which are insoluble in oil and thus moreamenable to physical separation. Even the addition of small amountscorresponding to about 700 lecitase units/kg oil results in a residual Pconcentration of less than 10 ppm, so that chemical refining isreplaceable by physical refining, eliminating the necessity forneutralization, soapstock splitting, and wastewater treatment.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by EnzyMax, Dahlke,Klaus, Dept. G-PDO, Lurgi Ol-Gas, Chemie, GmbH, Frankfurt, Germany.Oleagineux, Corps Gras, Lipides (1997), 4(1), 55-57. This exemplaryprocess is a degumming process for the physical refining of almost anykind of oil. By an enzymatic-catalyzed hydrolysis, phosphatides areconverted to water-soluble lysophosphatides which are separated from theoil by centrifugation. The residual phosphorus content in theenzymatically degummed oil can be as low as 2 ppm P.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Cleenewerck, et al.,N.V. Vamo Mills, Izegem, Belg. Fett Wissenschaft Technologie (1992),94:317-22; and, Clausen, Kim; Nielsen, Munk. Novozymes A/S, Den. DanskKemi (2002) 83(2):24-27. The phospholipases and methods of the inventioncan incorporate the pre-refining of vegetable oils with acids asdescribed, e.g., by Nilsson-Johansson, et al., Fats Oils Div.,Alfa-Laval Food Eng. AB, Tumba, Swed. Fett Wissenschaft Technologie(1988), 90(11), 447-51; and, Munch, Ernst W. Cereol Deutschland GmbH,Mannheim, Germany. Editor(s): Wilson, Richard F. Proceedings of theWorld Conference on Oilseed Processing Utilization, Cancun, MX, Nov.12-17, (2001), Meeting Date 2000, 17-20.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Jerzewska, et al.,Inst. Przemyslu Miesnego i Tluszczowego, Warsaw, Pol., Tluszcze Jadalne(2001), 36(3/4), 97-110. In this process of the invention, enzymaticdegumming of hydrated low-erucic acid rapeseed oil is by use of aphospholipase A2 of the invention. The enzyme can catalyze thehydrolysis of fatty acid ester linkages to the central carbon atom ofthe glycerol moiety in phospholipids. It can hydrolyze non-hydratablephospholipids to their corresponding hydratable lyso-compounds. With anonpurified enzyme preparation, better results can be achieved with theaddition of 2% preparation for 4 hours (87% P removal).

In another exemplary process of the invention for oil degumming (or anoil degumming process using an enzyme of the invention), an acidicpolymer, e.g., an alginate or pectin, is added. In this oil degummingprocess of the invention, an acidic polymer (e.g. alginic acid or pectinor a more soluble salt form) is added to the crude oil with a low amountof water (e.g., in a range of between about 0.5 to 5%). In this aspect,the acidic polymers can reduce and/or disrupt phospholipid-metalcomplexes by binding calcium and/or magnesium in the crude oil, therebyimproving the solubility of nonhydratable phospholipids. In alternativeaspects, these phospholipids will move to the oil/water interface orenter the aqueous phase and either be converted to diacylglycerol andthe corresponding side chain or the intact phospholipid will be removedby subsequent centrifugation as a component of the heavy phase. Thepresence of the acidic polymer in the aqueous phase can also increasethe density of the aqueous phase and result in an improved separation ofthe heavy phase from the oil (light) phase.

One exemplary process of the invention for oil degumming (or an oildegumming process using an enzyme of the invention) alters thedeodorization procedure to get a diacylglycerol (DAG) fraction. Inalternative aspect, if necessary or desired, following enzyme-assisteddegumming, the deodorization conditions (temperature, pressure,configuration of the distillation apparatus) can be modified with thegoal of improving the separation of the free fatty acids (FFA) from thediacylglycerol/triacylglycerol fraction or further modified to separatethe diacylglycerol from the triacylglycerol fraction. As a result ofthese modifications, using this method of the invention, it is possibleto obtain food grade FFA and diacylglycerol if an enzyme of theinvention (e.g., a phosphatase, or, a PLC or a combination of PLC andphosphatases) are used to degum edible oil in a physical refiningprocess.

In various aspects, practicing the methods of the invention as describedherein (or using the enzymes of the invention), have advantages such as:decrease or eliminate solvent and solvent recovery; lower capital costs;decrease downstream refining costs, decrease chemical usage, equipment,process time, energy (heat) and water usage/wastewater generation;produce higher quality oil; expeller pressed oil may be used withoutrefining in some cooking and sauteing applications (this pressed oil mayhave superior stability, color and odor characteristics and hightocopherol content); produce higher quality meal; produce a lower fatcontent in meal (currently, meal coming out of mechanical press causesdigestion problems in ruminants); produce improved nutritionalattributes—reduced levels of glucosinolates, tannins, sinapine, phyticacid (as described, e.g., in Technology and Solvents for ExtractingOilseeds and Nonpetroleum Oils, AOCS 1997).

In one aspect, the invention provides methods for refining vegetableoils (e.g., soybean oil, corn oil, cottonseed oil, palm oil, peanut oil,rapeseed oil, safflower oil, sunflower seed oil, sesame seed oil, ricebran oil, coconut oil or canola oil) and their byproducts, and processesfor deodorizing lecithin, for example, as described in U.S. Pat. No.6,172,248, or 6,172,247, wherein the methods comprise use of at leastone enzyme of the invention, e.g., a phospholipase C of the invention.Thus, the invention provides lecithin and vegetable oils comprising atleast one enzyme of the invention. In an exemplary organic acid refiningprocess, vegetable oil is combined with a dilute aqueous organic acidsolution and subjected to high shear to finely disperse the acidsolution in the oil. The resulting acid-and-oil mixture is mixed at lowshear for a time sufficient to sequester contaminants into a hydratedimpurities phase, producing a purified vegetable oil phase. In thisexemplary process, a mixer or recycle system (e.g., recycle water tank)and/or a phosphatide or lecithin storage tank can be used, e.g., asdescribed in U.S. Pat. No. 4,240,972, 4,049,686, 6,172,247 or 6,172,248.These processes can be conducted as a batch or continuous process. Crudeor degummed vegetable oil can be supplied from a storage tank (e.g.,through a pump) and can be heated. The vegetable oil to be purified canbe either crude or “degummed” oil.

In one aspect, phosphatidylinositol-PLC (PI-PLC) enzymes of theinvention are used for vegetable oil degumming. PI-PLC enzymes of theinvention can be used alone or in combination with other enzymes (forinstance PLC, PLD, phosphatase enzymes of the invention) to improve oilyield during the degumming of vegetable oils (including soybean, canola,and sunflower). The PI-PLC may preferentially convertphosphatidylinositol to 1,2-diacylglycerol (DAG) and phosphoinositol butit may also demonstrate activity on other phospholipids includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, orphosphatidic acid, or a combination thereof. The improvement in yieldwill be realized as an increase in the amount of DAG in theenzyme-treated vegetable oil and an increase in neutral oil, due to adecrease in the amount of oil entrained in the smaller gum fraction thatresults from enzyme treatment of the vegetable oil.

Enzymatic Processing of Oilseeds

The invention provides compositions (e.g., enzymes) and methods forenzymatic processing of oilseeds, including soybean, canola, coconut,avocado and olive paste. In one aspect, these processes of the inventioncan increase the oil yield and to improve the nutritional quality of theobtained meals. In some aspects, enzymatic processing of oilseeds usingthe enzymes and methods of the invention will provide economical andenvironmental benefits, as well as alternative technologies for oilextraction and processing food for human and animal consumption. Inalternative aspects, the processes of the invention comprise use ofphospholipases of the invention, other phospholipases, proteases,phosphatases, phytases, xylanases, amylases (e.g., α-amylases),glucanases (e.g., β-glucanases), polygalacturonases, galactolipases,cellulases, hemicellulases, pectinases and other plant cell walldegrading enzymes, as well as mixed enzyme preparations and celllysates.

In alternative aspects, the processes of the invention can be practicedin conjunction with other processes, e.g., enzymatic treatments, e.g.,with carbohydrases, including cellulase, hemicellulase and other sidedegrading activities, or, chemical processes, e.g., hexane extraction ofsoybean oil. The enzymatic treatment can increase the oil extractabilityby 8-10% when the enzymatic treatment is carried out prior to thesolvent extraction.

In alternative aspects, the processes of the invention can be practicedwith aqueous extraction processes. The aqueous extraction methods can beenvironmentally cleaner alternative technologies for oil extraction. Lowextraction yields of aqueous process can be overcome by using enzymesthat hydrolyze the structural polysaccharides forming the cell wall ofoilseeds, or that hydrolyze the proteins which form the cell and lipidbody membranes, e.g., utilizing digestions comprising cellulase,hemicellulase, and/or protopectinase for extraction of oil from soybeancells. In one aspect, methods are practiced with an enzyme of theinvention as described by Kasai (2003) J. Agric. Food Chem.51:6217-6222, who reported that the most effective enzyme to digest thecell wall was cellulase.

In one aspect, proteases are used in combination with the methods of theinvention. The combined effect of operational variables and enzymeactivity of protease and cellulase on oil and protein extraction yieldscombined with other process parameters, such as enzyme concentration,time of hydrolysis, particle size and solid-to-liquid ratio has beenevaluated. In one aspect, methods are practiced with an enzyme of theinvention as described by Rosenthal (2001) Enzyme and Microb. Tech.28:499-509, who reported that use of protease can result insignificantly higher yields of oil and protein over the control whenheat treated flour is used.

In one aspect, complete protein, pectin, and hemicellulose extractionare used in combination with the methods of the invention. The plantcell consists of a series of polysaccharides often associated with orreplaced by proteins or phenolic compounds. Most of these carbohydratesare only partially digested or poorly utilized by the digestive enzymes.The disruption of these structures through processing or degradingenzymes can improve their nutrient availability. In one aspect, methodsare practiced with an enzyme of the invention as described by Ouhida(2002) J. Agric. Food Chem. 50:1933-1938, who reported that asignificant degradation of the soybean cell wall cellulose (up to 20%)has been achieved after complete protein, pectin, and hemicelluloseextraction.

In one aspect, the methods of the invention further compriseincorporation of various enzymatic treatments in the treatment of seeds,e.g., canola seeds, these treatments comprising use of proteases,cellulases, and hemicellulases (in various combinations with each otherand with one or more enzymes of the invention). For example, the methodscan comprise enzymatic treatments of canola seeds at 20 to 40 moistureduring the incubation with enzymes prior to a conventional process; asdescribed, e.g., by Sosulski (1990) Proc. Can. Inst. Food Sci. Technol.3:656. The methods of the invention can further comprise incorporationof proteases, α-amylases, polygalacturonases (in various combinationswith each other and with one or more enzymes of the invention) tohydrolyze cellular material in coconut meal and release the coconut oil,which can be recovered by centrifugation, as described, e.g., by McGlone(1986) J. of Food Sci. 51:695-697. The methods of the invention canfurther comprise incorporation of pectinases, α-amylases, proteases,cellulases in different combinations (with each other and with one ormore enzymes of the invention) to result in significant yieldimprovement (˜70% in the best case) during enzymatic extraction ofavocado oil, as described, e.g., by Buenrostro (1986) Biotech. Letters8(7):505-506. In processes of the invention for olive oil extraction,olive paste is treated with cellulase, hemicellulase, poligalacturonase,pectin-methyltransferase, protease and their combinations (with eachother and with one or more enzymes of the invention), as described,e.g., by Montedoro (1976) Acta Vitamin. Enzymol. (Milano) 30:13.

Purification of Phytosterols from Vegetable Oils

The invention provides methods for purification of phytosterols andtriterpenes, or plant sterols, from vegetable oils. Phytosterols thatcan be purified using phospholipases and methods of the inventioninclude β-sitosterol, campesterol, stigmasterol, stigmastanol,β-sitostanol, sitostanol, desmosterol, chalinasterol, poriferasterol,clionasterol and brassicasterol. Plant sterols are importantagricultural products for health and nutritional industries. Thus,phospholipases and methods of the invention are used to make emulsifiersfor cosmetic manufacturers and steroidal intermediates and precursorsfor the production of hormone pharmaceuticals. Phospholipases andmethods of the invention are used to make (e.g., purify) analogs ofphytosterols and their esters for use as cholesterol-lowering agentswith cardiologic health benefits. Phospholipases and methods of theinvention are used to purify plant sterols to reduce serum cholesterollevels by inhibiting cholesterol absorption in the intestinal lumen.Phospholipases and methods of the invention are used to purify plantsterols that have immunomodulating properties at extremely lowconcentrations, including enhanced cellular response of T lymphocytesand cytotoxic ability of natural killer cells against a cancer cellline. Phospholipases and methods of the invention are used to purifyplant sterols for the treatment of pulmonary tuberculosis, rheumatoidarthritis, management of HIV-infested patients and inhibition of immunestress, e.g., in marathon runners.

Phospholipases and methods of the invention are used to purify sterolcomponents present in the sterol fractions of commodity vegetable oils(e.g., coconut, canola, cocoa butter, corn, cottonseed, linseed, olive,palm, peanut, rice bran, safflower, sesame, soybean, sunflower oils),such as sitosterol (40.2-92.3%), campesterol (2.6-38.6%), stigmasterol(0-31%) and 5-avenasterol (1.5-29%).

Methods of the invention can incorporate isolation of plant-derivedsterols in oil seeds by solvent extraction with chloroform-methanol,hexane, methylene chloride, or acetone, followed by saponification andchromatographic purification for obtaining enriched total sterols.Alternatively, the plant samples can be extracted by supercritical fluidextraction with supercritical carbon dioxide to obtain total lipidextracts from which sterols can be enriched and isolated. For subsequentcharacterization and quantification of sterol compounds, the crudeisolate can be purified and separated by a wide variety ofchromatographic techniques including column chromatography (CC), gaschromatography, thin-layer chromatography (TLC), normal phasehigh-performance liquid chromatography (HPLC), reversed-phase HPLC andcapillary electrochromatography. Of all chromatographic isolation andseparation techniques, CC and TLC procedures employ the most accessible,affordable and suitable for sample clean up, purification, qualitativeassays and preliminary estimates of the sterols in test samples.

Phytosterols are lost in the vegetable oils lost as byproducts duringedible oil refining processes. Phospholipases and methods of theinvention use phytosterols isolated from such byproducts to makephytosterol-enriched products isolated from such byproducts. Phytosterolisolation and purification methods of the invention can incorporate oilprocessing industry byproducts and can comprise operations such asmolecular distillation, liquid-liquid extraction and crystallization.

Methods of the invention can incorporate processes for the extraction oflipids to extract phytosterols. For example, methods of the inventioncan use nonpolar solvents as hexane (commonly used to extract most typesof vegetable oils) quantitatively to extract free phytosterols andphytosteryl fatty-acid esters. Steryl glycosides and fatty-acylatedsteryl glycosides are only partially extracted with hexane, andincreasing polarity of the solvent gave higher percentage of extraction.One procedure that can be used is the Bligh and Dyer chloroform-methanolmethod for extraction of all sterol lipid classes, includingphospholipids. One exemplary method to both qualitatively separate andquantitatively analyze phytosterol lipid classes comprises injection ofthe lipid extract into HPLC system.

Phospholipases and methods of the invention can be used to removesterols from fats and oils, as described, e.g., in U.S. Pat. No.6,303,803. This is a method for reducing sterol content ofsterol-containing fats and oils. It is an efficient and cost effectiveprocess based on the affinity of cholesterol and other sterols foramphipathic molecules that form hydrophobic, fluid bilayers, such asphospholipid bilayers. Aggregates of phospholipids are contacted with,for example, a sterol-containing fat or oil in an aqueous environmentand then mixed. The molecular structure of this aggregated phospholipidmixture has a high affinity for cholesterol and other sterols, and canselectively remove such molecules from fats and oils. The aqueousseparation mixture is mixed for a time sufficient to selectively reducethe sterol content of the fat/oil product through partitioning of thesterol into the portion of phospholipid aggregates. The sterol-reducedfat or oil is separated from the aqueous separation mixture.Alternatively, the correspondingly sterol-enriched fraction also may beisolated from the aqueous separation mixture. These steps can beperformed at ambient temperatures, costs involved in heating areminimized, as is the possibility of thermal degradation of the product.Additionally, a minimal amount of equipment is required, and since allrequired materials are food grade, the methods require no specialprecautions regarding handling, waste disposal, or contamination of thefinal product(s).

Phospholipases and methods of the invention can be used to removesterols from fats and oils, as described, e.g., in U.S. Pat. No.5,880,300. Phospholipid aggregates are contacted with, for example, asterol-containing fat or oil in an aqueous environment and then mixed.Following adequate mixing, the sterol-reduced fat or oil is separatedfrom the aqueous separation mixture. Alternatively, the correspondinglysterol-enriched phospholipid also may be isolated from the aqueousseparation mixture. Plant (e.g., vegetable) oils contain plant sterols(phytosterols) that also may be removed using the methods of the presentinvention. This method is applicable to a fat/oil product at any stageof a commercial processing cycle. For example, the process of theinvention may be applied to refined, bleached and deodorized oils (“RBDoils”), or to any stage of processing prior to attainment of RBD status.Although RBD oil may have an altered density compared to pre-RBD oil,the processes of the are readily adapted to either RBD or pre-RBD oils,or to various other fat/oil products, by variation of phospholipidcontent, phospholipid composition, phospholipid:water ratios,temperature, pressure, mixing conditions, and separation conditions asdescribed below.

Alternatively, the enzymes and methods of the invention can be used toisolate phytosterols or other sterols at intermediate steps in oilprocessing. For example, it is known that phytosterols are lost duringdeodorization of plant oils. A sterol-containing distillate fractionfrom, for example, an intermediate stage of processing can be subjectedto the sterol-extraction procedures described above. This provides asterol-enriched lecithin or other phospholipid material that can befurther processed in order to recover the extracted sterols.

Detergent Compositions

The invention provides detergent compositions comprising one or morephospholipase of the invention, and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;6,365,561; 6,380,147. The detergent compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The invention also provides methodscapable of a rapid removal of gross food soils, films of food residueand other minor food compositions using these detergent compositions.Phospholipases of the invention can facilitate the removal of stains bymeans of catalytic hydrolysis of phospholipids. Phospholipases of theinvention can be used in dishwashing detergents in textile launderingdetergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof phospholipase present in the final solution ranges from about 0.001mg to 0.5 mg per gram of the detergent composition. The particularenzyme chosen for use in the process and products of this inventiondepends upon the conditions of final utility, including the physicalproduct form, use pH, use temperature, and soil types to be degraded oraltered. The enzyme can be chosen to provide optimum activity andstability for any given set of utility conditions. In one aspect, thepolypeptides of the present invention are active in the pH ranges offrom about 4 to about 12 and in the temperature range of from about 20°C. to about 95° C. The detergents of the invention can comprisecationic, semi-polar nonionic or zwitterionic surfactants; or, mixturesthereof.

Phospholipases of the present invention can be formulated into powderedand liquid detergents having pH between 4.0 and 12.0 at levels of about0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as known proteases,cellulases, lipases or endoglycosidases, as well as builders andstabilizers. The addition of phospholipases of the invention toconventional cleaning compositions does not create any special uselimitation. In other words, any temperature and pH suitable for thedetergent is also suitable for the present compositions as long as thepH is within the above range, and the temperature is below the describedenzyme's denaturing temperature. In addition, the polypeptides of theinvention can be used in a cleaning composition without detergents,again either alone or in combination with builders and stabilizers.

The present invention provides cleaning or disinfecting compositionsincluding detergent and/or disinfecting compositions for cleaning and/ordisinfecting hard surfaces, detergent compositions for cleaning and/ordisinfecting fabrics, dishwashing compositions, oral cleaningcompositions, denture cleaning compositions, and/or contact lenscleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a phospholipase of the inventionunder conditions sufficient for washing. A phospholipase of theinvention may be included as a detergent additive. The detergentcomposition of the invention may, for example, be formulated as a handor machine laundry detergent composition comprising a phospholipase ofthe invention. A laundry additive suitable for pre-treatment of stainedfabrics can comprise a phospholipase of the invention. A fabric softenercomposition can comprise a phospholipase of the invention.Alternatively, a phospholipase of the invention can be formulated as adetergent composition for use in general household hard surface cleaningoperations. In alternative aspects, detergent additives and detergentcompositions of the invention may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, another phospholipase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase. The properties of the enzyme(s) of the invention are chosento be compatible with the selected detergent (i.e. pH-optimum,compatibility with other enzymatic and non-enzymatic ingredients, etc.)and the enzyme(s) is present in effective amounts. In one aspect,phospholipase enzymes of the invention are used to remove malodorousmaterials from fabrics. Various detergent compositions and methods formaking them that can be used in practicing the invention are describedin, e.g., U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038;6,309,871; 6,204,232; 6,197,070; 5,856,164.

Waste Treatment

The phospholipases of the invention can be used in waste treatment. Inone aspect, the invention provides a solid waste digestion process usingphospholipases of the invention. The methods can comprise reducing themass and volume of substantially untreated solid waste. Solid waste canbe treated with an enzymatic digestive process in the presence of anenzymatic solution (including phospholipases of the invention) at acontrolled temperature. The solid waste can be converted into aliquefied waste and any residual solid waste. The resulting liquefiedwaste can be separated from said any residual solidified waste. Seee.g., U.S. Pat. No. 5,709,796.

Detoxification

The phospholipases (e.g., PLCs, patatins of the invention) can be usedin detoxification processes, e.g., for the detoxification of endotoxins,e.g., compositions comprising lipopolysaccharides (LPS), and, theinvention provides detoxification processes using at least one enzyme ofthe invention, e.g., a patatin having a sequence as set forth in SEQ IDNO:12 (encoded by SEQ ID NO:11), SEQ ID NO:14 (encoded by SEQ ID NO:13), SEQ ID NO:18 (encoded by SEQ ID NO:17), SEQ ID NO:26 (encoded bySEQ ID NO:25), SEQ ID NO:28 (encoded by SEQ ID NO:27), SEQ ID NO:34(encoded by SEQ ID NO:33), SEQ ID NO:36 (encoded by SEQ ID NO:35), SEQID NO:44 (encoded by SEQ ID NO:43), SEQ ID NO:46 (encoded by SEQ IDNO:45), SEQ ID NO:56 (encoded by SEQ ID NO:55), SEQ ID NO:60 (encoded bySEQ ID NO:59), SEQ ID NO:66 (encoded by SEQ ID NO:65), SEQ ID NO:72(encoded by SEQ ID NO:71), SEQ ID NO:78 (encoded by SEQ ID NO:77), SEQID NO:87 (encoded by SEQ ID NO:86), SEQ ID NO:88 (encoded by SEQ IDNO:87), SEQ ID NO:92 (encoded by SEQ ID NO:91), SEQ ID NO:96 (encoded bySEQ ID NO:95), SEQ ID NO:100 (encoded by SEQ ID NO:99), SEQ ID NO:104(encoded by SEQ ID NO:103), SEQ ID NO:126 (encoded by SEQ ID NO:125),SEQ ID NO:128 (encoded by SEQ ID NO:127), SEQ ID NO:132 (encoded by SEQID NO:131), SEQ ID NO:134 (encoded by SEQ ID NO:133), SEQ ID NO:136(encoded by SEQ ID NO:135), or SEQ ID NO:138 (encoded by SEQ ID NO:137).In one aspect, a phospholipase of the invention is used to detoxify alipopolysaccharide (LPS). In one aspect, this detoxification is bydeacylation of 2′ and/or 3′ fatty acid chains from lipid A. In oneaspect, a phospholipase (e.g., a PLC, a patatin) of the invention isused to hydrolyze a 2′-lauroyl and/or a 3′-myristoyl chain from a lipid,e.g., a lipid A (e.g., from a bacterial endotoxin). In one aspect, theprocess of the invention is used to destroy an endotoxin, e.g., a toxinfrom a gram negative bacteria, as from E. coli. In one aspect, aphospholipase (e.g., a PLC, a patatin) of the invention is used toameliorate the effects of toxin poisoning (e.g., from an on-going gramnegative infection), or, to prophylactically to prevent the effects ofendotoxin during an infection (e.g., an infection in an animal or ahuman). Accordingly, the invention provides a pharmaceutical compositioncomprising a phospholipase (e.g., a PLC, a patatin) of the invention,and method using a hydrolase of the invention, for the amelioration orprevention of lipopolysaccharide (LPS) toxic effects, e.g., duringsepsis.

Processing Foods

The phospholipases of the invention can be used to process foods, e.g.,to change their stability, shelf-life, flavor, texture, improve on theirnutritional status, and the like. For example, in one aspect,phospholipases of the invention are used to generate acidicphospholipids for controlling bitter taste in foods.

In one aspect, the invention provides cheese-making processes usingphospholipases of the invention (and, thus, the invention also providescheeses comprising phospholipases of the invention). In one aspect, theenzymes of the invention (e.g., phospholipase A, lysophospholipase or acombination thereof) are used to process cheeses for flavor enhancement,to increase yield and/or for “stabilizing” cheeses, e.g., by reducingthe tendency for “oil-off,” or, in one aspect, the enzymes of theinvention are used to produce cheese from cheese milk. These processesof the invention can incorporate any method or protocol, e.g., asdescribed, e.g., in U.S. Pat. Nos. 6,551,635, and 6,399,121, WO03/070013, WO 00/054601. For example, in one aspect, the phospholipasesof the invention are used to stabilize fat emulsion in milk ormilk-comprising compositions, e.g. cream, and are used to stabilize milkcompositions, e.g. for the manufacturing of creams or cream liquors. Inone aspect, the invention provides a process for enhancing the favor ofa cheese using at least one enzyme of the invention, the processcomprising incubating a protein, a fat and a protease and a lipase in anaqueous medium under conditions that produce an enhanced cheese flavor(e.g., reduced bitterness), e.g., as described in WO 99/66805. In oneaspect, phospholipases of the invention are used to enhance flavor in acheese (e.g., a curd) by mixing with water, a protease, and a lipase (ofthe invention) at an elevated temperature, e.g., between about 75° C. to95° C., as described, e.g., in U.S. Pat. No. 4,752,483. In one aspect,phospholipases of the invention are used to accelerate cheese aging byadding an enzyme of the invention (e.g., a lipase or a phospholipase) toa cheese (e.g., a cheese milk) before adding a coagulant to the milk,or, adding an enzyme of the invention to a curd with salt beforepressing, e.g., as described, e.g., in U.S. Pat. No. 4,707,364. In oneaspect, a lipase of the invention is used degrade a triglyceride in milkfat to liberate free fatty acids, resulting in flavor enhancement. Aprotease also can be used in any of these processes of the invention,see, e.g., Brindisi (2001) J. of Food Sci. 66:1100-1107. In anotheraspect, a combination of esterases, lipases, phospholipases and/orproteases can be used in these or any process of the invention.

In one aspect, a phospholipase of the invention is used to reduce thecontent of phosphorus components in a food, e.g., an oil, such as avegetable oil having a high non-hydratable phosphorus content, e.g., asdescribed in WO 98/26057.

Other Uses for the Phospholipases of the Invention

The phospholipases of the invention can also be used to study thephosphoinositide (PI) signaling system; in the diagnosis, prognosis anddevelopment of treatments for bipolar disorders (see, e.g., Pandey(2002) Neuropsychopharmacology 26:216-228); as antioxidants; as modifiedphospholipids; as foaming and gelation agents; to generate angiogeniclipids for vascularizing tissues; to identify phospholipase, e.g., PLA,PLB, PLC, PLD and/or patatin modulators (agonists or antagonists), e.g.,inhibitors for use as anti-neoplastics, anti-inflammatory and asanalgesic agents. They can be used to generate acidic phospholipids forcontrolling the bitter taste in food and pharmaceuticals. They can beused in fat purification. They can be used to identify peptidesinhibitors for the treatment of viral, inflammatory, allergic andcardiovascular diseases. They can be used to make vaccines. They can beused to make polyunsaturated fatty acid glycerides andphosphatidylglycerols.

The phospholipases of the invention, for example PLA and PLC enzymes,are used to generate immunotoxins and various therapeutics foranti-cancer treatments.

The phospholipases of the invention can be used in conjunction withother enzymes for decoloring (i.e. chlorophyll removal) and indetergents (see above), e.g., in conjunction with other enzymes (e.g.,lipases, proteases, esterases, phosphatases). For example, in anyinstance where a PLC is used, a PLD and a phosphatase may be used incombination, to produce the same result as a PLC alone.

The following table summaries several exemplary processes andformulations of the invention:

Exemplary Process of the invention Purpose Chemical usage in PLC oildegumming No use of acid Chemical elimination No use of caustic Chemicalelimination Range of acid and caustic use (no excess Chemicalreduction/degumming process to excess) alternative embodiment Othertypes of acid and caustic Degumming process alternative embodimentsImpact of water in PLC oil degumming Use of silica gel Replacement ofwater wash step Use of water drying agent Elimination of water in finalproduct Impact of lower water during caustic Elimination of water infinal product treatment Minimal water content (<5%) Elimination of waterin final product Maximal water content (>5%) Process alternativeHumidity profiles on PLC degumming Degumming process alternativeembodiment Oil dependence on water content for PLC Degumming processalternative degumming embodiment In situ removal of free fatty acids,FFAs Addition of FFA chelating agent- Degumming process alternativeembodiment; improves conditions in oil from spoilt beans Impact ofmixing regimen on PLC oil degumming PLC degumming with minimal mixingProtection of enzyme from mixing induced denaturation, energy savingsPLC degumming with initial shear Degumming process alternative mixing,followed by paddle mixing embodiment Order of addition of chemicalsOrder of addition: enzyme-water followed Allow the PLC to work beforeexposure to by acid then caustic acid and or caustic, causing potentialpH or metal chelation PLC inactivation PLC oil degumming processalternative embodiments for temperature and time Enzyme treatment step(time): <60 min, Degumming process alternative preferably <30 minembodiment Enzyme treatment step (temperature): 50- Degumming processalternative 70° C., possibly <50° C. (e.g. RT) embodiment Benefits fromPLC oil degumming Producing soapstock with minimized PL Degummingprocess alternative content and enriched in water soluble embodimentphosphate esters Reduced neutral oil in gum through use of Degummingprocess alternative PLC embodiment Process of generating increase of DAGin Degumming process alternative vegetable oils (for ex, 1,3-DAG)embodiment Benefits of using increased DAG Exemplary Product benefitvegetable oils with other oils for health benefits Investigate degummingprocess that Degumming process alternative leaves no PLC activity in oilembodiment/regulatory improvement Investigate degumming process thatDegumming process alternative leaves no detectable PLC protein in oilembodiment/regulatory improvement Use of an enzyme to produce DAG fromExemplary Product benefit lecithin gum mass Use of PLC with specialtyoils (PA, PI Exemplary Product benefit enriched) Use of PA/PI specificenzymes (e.g. Degumming process alternative 596ES2/PI specific)embodiment Use of PA/PI specific enzymes (e.g. Degumming processalternative 596ES2/PI specific) + PC/PE specific embodiment enzymes;impact of order of addition Batch or continuous process Degummingprocess alternative embodiment Use of resuspended PLC treated gum forDegumming process alternative further oil degumming operationsembodiment Mass balance for DAG, FFA, P, metals, Degumming processalternative neutral oil in gum embodiment Miscellaneous Addition of PLCto flaked oil seed kernels Process alternative embodiment beforeextrusion Small scale degumming assay Degumming process alternativeembodiment Use of other enzymes to reduce gum mass Degumming processalternative (e.g., PYROLASE ™ enzyme, embodiment chlorophyllase,peroxidase, lipase, laccase, mannanase, protease, lactase, amylase, etc.or combinations thereof) Use of compound to better facilitate Degummingprocess alternative oil/gum separation embodiment Harden gum from PLCtreated oil Degumming process alternative embodimentGlycosylated/deglycosylated variants of Degumming process alternativephospholipase embodiment Exemplary Liquid formulation for stability Useof compounds to increase the stability Stabilization of enzyme formaximum of PLC at different pH and temp, ranges DAG production, possiblyfor altering (polyols, salts, metals . . . ) substrate specificity ordirecting product formation towards the 1,3-DAG type Stabilization ofenzyme for maximum Use of a hydrophobic delivery system for DAGproduction, possibly for altering PLC (liposomes, hydrated enzyme insubstrate specificity or directing product refined oil droplets)formation towards the 1,3-DAG type Solid formulation for stability Useof different PLC, phospholipase Stabilization of the enzyme(s) and easeof carrier systems (immobilization resins, separation of the enzyme fromthe oil or porous matrices, gels, granules, powders, gum phase afterdegumming; recyclability tablets, vesicles/micelles, encapsulates, ofthe enzyme preparation; physical structured liquids, etc) to stabilizeseparation of the enzyme phase during oil phospholipase and co-enzymesprocessing; attack of PI/PA by PLC Use of degumming waste materials (gumCost reduction of formulation ingredient, components, seed hulls) forPLC better miscibility of enzyme with oil, formulationthermostabilization of enzyme Exemplary Formulation and processes foractivity boost Use of chemical or enzyme to help Faster reactiontime/degumming disperse the enzyme better in oil (e.g. process/reductionof chemical usage effervescent matrix, etc) Re-use of gums/enzyme forfurther Recyclability of enzyme degumming reactions Use of formulationsthat enhance the Faster reaction time/degumming segregation or enzymecapture of PLs for process/reduction of chemical usage hydrolysis Use ofmultiple formulations to Versatility of process; different enzymesaccommodate PLCs with different PL may require different formulations ormay specificities be added at different stages in the process Use ofmultiple formulations to prevent Protection of PLC activities in amulti- inactivation of one PLC by a component enzyme format embodimentin the prep of another PLC with a different substrate specificity Use ofmultiple formulations to prevent Protection of PLC activity in a multi-inactivation of one PLC by a component enzyme format embodiment in theprep of another enzyme (hydrolase, oxidase) Use of intermittent causticadditions as in Protection of enzyme from mixing time released causticaddition formulation induced denaturation, energy savings

Inactivating and Modulating Activity of Enzymes by Glycosylation

This invention provides methods comprising use of recombinant technologyto make and expressing enzymes or other proteins with biologicalactivity, e.g., noxious or toxic enzymes, (wherein the enzymes or otherproteins are not normally glycosylated) in an inactive or less active,but re-activatable, form. The method comprises adding one or moreglycosylation sites (e.g., N-linked or O-linked glycosylation) into theenzymes or other proteins with biological activity (e.g., an enzyme ofthe present invention) by engineering a coding sequence incorporatingthe new glycosylation site(s); expressing the variant coding sequencesin eukaryotic cells or an equivalent engineered or in vitro systemcapable of post-translational glycosylation. For example, the 3 aminoacid sequence NXS/T is the site for glycosylation in eukaryotic cells,prokaryotic cells do not do this. Thus, the invention comprises addingat least one 3 amino acid sequence NXS/T to the protein such that itsactivity is decreased or inactivated because of post-translationalglycosylation.

The glycosylation can result in 2 molecules of N-acetyl glucosamine(NGlucNac) being added to the N residue. Subsequent additions can beorganism specific. In most species mannose (Mann) sugars are then addedonto the NGlucNac, with the number Mann residues ranging from 10 to 100.Sialic acid can also be added in some species. In Pichia after theNGlucNac is added, 10 to 25 Mann residues can be added.

These methods comprise using any deglycosylating enzyme or set ofenzymes, many of which can have been identified and/or are commerciallyavailable. For example, the endoglycosidase H enzyme cleaves at the lastNGlucNac leaving one NClucNac still attached to the N residue. ThePNGaseF enzyme cleaves off all of the sugars and converts the amino sidechain of the N residue into a hydroxyl group resulting in the N aminoacid becoming an aspartate (D) amino acid in the enzyme. Thus, themethods comprise using endoglycosidase H and/or PNGaseF or equivalentenzymes in vivo or in vitro to re-activate partially or completely theengineered “temporarily inactivated” proteins.

The method comprises targeting the enzymes or other polypeptides to thehost secretory pathway so that the enzymes will be glycosylated. The newglycosylation sites are designed such that glycosylation inactivates theenzyme or modifies its activity, e.g., decreases it activity or otherotherwise modifies activity, such as blocks a substrate binding site.Because the enzyme is inactive or less active, noxious or toxic enzymescould be expressed at higher levels since the negative effects of theiractivity are no longer a limitation to how much of the protein canaccumulate in the host cells. The inactive, glycosylated enzyme can bere-activated (partially or completely) by removing the sugars, e.g.,using commercially available deglycosylating enzymes, for example, byremoving the sugars in vitro, or removing the sugars in vivo using wholecell engineering approaches.

In one aspect, a eukaryotic glycosylation target site such as NXS/T isadded to any protein, for example, an enzyme of the invention. Thisenables one skilled in the art to add glycosylation sites to a proteinof interest with the expectation of converting that protein into onethat is temporarily inactive when that protein is glycosylated byexpressing that protein in a eukaryotic host cell and targeting theprotein to the host cell's secretory pathway.

Thus, the invention provides methods for the production of enzymes thatnormally are too noxious or toxic to be tolerated in large amounts by ahost cell. The effect can temporary as it is possible to regenerate theactive enzyme (by deglycosylation, e.g., by post-translationalmodification/deglycosylation) for future work requiring an activeenzyme.

In one aspect, the invention provides methods for making and expressinga protein having a biological activity whose activity is temporarilyinactivated by glycosylation comprising: (a) providing a nucleic acidencoding a protein having a biological activity, wherein the protein isnot naturally glycosylated; (b) inserting at least one glycosylationmotif coding sequence into the protein-encoding nucleic acid, whereinthe glycosylated form of the protein is inactive; (c) inserting atargeting sequence into the protein such that it is directed to a hostcell's secretory pathway, wherein the host cell is capable ofrecognizing the glycosylation motif and glycosylating the protein; and(d) expressing the modified nucleic acid in the host cell. In oneaspect, the method further comprises deglycosylating the expressed theprotein, thereby re-activating the activity of the protein, e.g., anenzyme, such as an enzyme of the invention. In one aspect, the host cellis a eukaryotic cell. In one aspect, the inactivated expressedrecombinant protein is re-activated in vitro by deglycosylation, eitherchemical or enzymatic.

Determining the placement of one or more glycosylation motifs totemporarily inactivate a protein involves only routine methods of makingvariant protein-encoding nucleic acids, e.g., by GSSM™, and routinescreening protocols, e.g., activity or binding assays.

An enzyme whose activity was detrimental to the host cell was renderedinactive because of glycosylation. Because it was inactive it couldaccumulate in much higher levels in the eukaryotic host cells. Becauseit was no longer active it could no longer able to exert its negativeeffects. The inactivation of the toxic enzyme was temporary becausedeglycosylating the enzyme using EndoH or PNGase F resulted in acomplete restoration of normal activity to the enzyme. A large amount ofthe glycosylated, inactive enzyme accumulated in the medium suggestingthat it was tolerated well by the host as the inactive form.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Blast Program used for Sequence Identify Profiling

This example describes an exemplary sequence identity program todetermine if a nucleic acid is within the scope of the invention. AnNCBI BLAST 2.2.2 program is used, default options to blastp. All defaultvalues were used except for the default filtering setting (i.e., allparameters set to default except filtering which is set to OFF); in itsplace a “−F F” setting is used, which disables filtering. Use of defaultfiltering often results in Karlin-Altschul violations due to shortlength of sequence. The default values used in this example:

-   -   “Filter for low complexity: ON    -   >Word Size: 3    -   >Matrix: Blosum62    -   >Gap Costs: Existence:11    -   >Extension: 1

Other default settings were: filter for low complexity OFF, word size of3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gapextension penalty of −1. The “−W” option was set to default to 0. Thismeans that, if not set, the word size defaults to 3 for proteins and 11for nucleotides. The settings read:

<<README.bls.txt>> >-------------------------------------------------------------------------- >blastall arguments: > > -p Program Name [String] > -d Database[String] > default = nr > -i Query File [File In] > default = stdin > -eExpectation value (E) [Real] > default =10.0 > -m alignment viewoptions: > 0 = pairwise, > 1 = query-anchored showing identities, > 2 =query-anchored no identities, > 3 = flat query-anchored, showidentities, > 4 = flat query-anchored, no identities, > 5 =query-anchored no identities and blunt ends, > 6 = flat query-anchored,no identities and blunt ends, > 7 = XML Blast output, > 8 = tabular, > 9tabular with comment lines [Integer] > default = 0 > -o BLAST reportOutput File [File Out] Optional > default = stdout > -F Filter querysequence (DUST with blastn, SEG with others) [String] > default = T > -GCost to open a gap (zero invokes default behavior) [Integer] > default =0 > -E Cost to extend a gap (zero invokes default behavior) [Integer] >default = 0 > -X X dropoff value for gapped alignment (in bits) (zeroinvokes > default behavior) [Integer] > default = 0 > -I Show GI's indeflines [T/F] > default = F > -q Penalty for a nucleotide mismatch(blastn only) [Integer] > default = −3 > -r Reward for a nucleotidematch (blastn only) [Integer] > default = 1 > -v Number of databasesequences to show one-line descriptions for (V) > [Integer] > default =500 > -b Number of database sequence to show alignments for (B)[Integer] > default = 250 > -f Threshold for extending hits, default ifzero [Integer] > default = 0 > -g Perform gapped alignment (notavailable with tblastx) [T/F] > default = T > -Q Query Genetic code touse [Integer] > default = 1 > -D DB Genetic code (for tblast[nx] only)[Integer] > default = 1 > -a Number of processors to use [Integer] >default = 1 > -O SeqAlign file [File Out] Optional > -J Believe thequery defline [T/F] > default = F > -M Matrix [String] > default =BLOSUM62 > -W Word size, default if zero [Integer] > default = 0 > -zEffective length of the database (use zero for the real size) >[String] > default = 0 > -K Number of best hits from a region to keep(off by default, if used a > value of 100 is recommended) [Integer] >default = 0 > -P 0 for multiple hits 1-pass, 1 for single hit 1-pass, 2for 2-pass > [Integer] > default = 0 > -Y Effective length of the searchspace (use zero for the real size) > [Real] > default = 0 > -S ;Querystrands to search against database (for blast[nx], and > tblastx). 3 isboth, 1 is top, 2 is bottom [Integer] > default = 3 > -T Produce HTMLoutput [T/F] > default = F > -l Restrict search of database to list ofGI's [String] Optional > -U Use lower case filtering of FASTA sequence[T/F] Optional > default = F > -y Dropoff (X) for blast extensions inbits (0.0 invokes default > behavior) [Real] > default = 0.0 > -Z Xdropoff value for final gapped alignment (in bits) [Integer] > default =0 > -R PSI-TBLASTN checkpoint file [File In] Optional > -n MegaBlastsearch [T/F] > default = F > -L Location on query sequence [String]Optional > -A Multiple Hits window size (zero for single hit algorithm)[Integer] > default = 40

Example 2 Simulation of PLC Mediated Degumming

This example describes the simulation of phospholipase C (PLC)-mediateddegumming.

Due to its poor solubility in water phosphatidylcholine (PC) wasoriginally dissolved in ethanol (100 mg/ml). For initial testing, astock solution of PC in 50 mM 3-morpholinopropanesulpholic acid or 60 mMcitric acid/NaOH at pH 6 was prepared. The PC stock solution (10 μl, 1μg/μl) was added to 5001 of refined soybean oil (2% water) in anEppendorf tube. To generate an emulsion the content of the tube wasmixed for 3 min by vortexing (see FIG. 5A). The oil and the water phasewere separated by centrifugation for 1 min at 13,000 rpm (FIG. 5B). Thereaction tubes were pre-incubated at the desired temperature (37° C.,50° C., or 60° C.) and 3 μl of PLC from Bacillus cereus (0.9 U/μl) wereadded to the water phase (FIG. 5C). The disappearance of PC was analyzedby TLC using chloroform/methanol/water (65:25:4) as a solvent system(see, e.g., Taguchi (1975) supra) and was visualized after exposure to12 vapor.

FIG. 5 schematically illustrates a model two-phase system for simulationof PLC-mediated degumming. FIG. 5A: Generation of emulsion by mixingcrude oil with 2% water to hydrate the contaminating phosphatides (P).FIG. 5B: The oil and water phases are separated after centrifugation andPLC is added to the water phase, which contains the precipitatedphosphatides (“gums”). The PLC hydrolysis takes place in the waterphase. FIG. 5C: The time course of the reaction is monitored bywithdrawing aliquots from the water phase and analyzing them by TLC.

Example 3 Expression of Phospholipases

This example describes the construction of a commercial productionstrain of the invention that can express multiple phospholipases(including enzymes of the invention). In order to produce a multi-enzymeformulation suitable for use in the degumming of food-grade vegetableoils (including soybean, canola, and sunflower), a recombinantexpression strain can be generated that expresses two differentphospholipase sequences in the same expression host. For example, thisstrain may be constructed to contain one or more copies of a PLC geneand one or more copies of a phosphatidylinositol-PLC gene. These genesmay exist on one plasmid, multiple plasmids, or the genes may beinserted into the genome of the expression host by homologousrecombination. When the genes are introduced by homologousrecombination, the genes may be introduced into a single site in thehost genome as a DNA expression cassette that contains one or morecopies of both genes. Alternatively, one or more copies of each gene maybe introduced into distinct sites in the host chromosome. The expressionof these two gene sequences could be driven by one type of promoter oreach gene sequence may be driven by an independent promoter. Dependingon the number of copies of each gene and the type of promoter, the finalstrain will express varying ratios of each active enzyme type. Theexpression strains can be constructed using any Bacillus (e.g., B.cereus) or Streptomyces, E. coli, S. pombe, P. pastoris, or othergram-negative, gram-positive, or yeast expression systems.

In one aspect, the invention provides a two-enzyme system for degummingof soybean oil, wherein at least one enzyme is an enzyme of theinvention. PLC plus PI-PLC produces more DAG than either enzyme alone.However both enzymes produce more DAG than a no enzyme control sample.In one aspect, reaction conditions comprise 1 milliliter soybean oil,˜0.4% initial moisture in the oil before any additions, 50° C., 0.2%Citric acid neutralized with 2.75M NaOH, 10 U PLC, 15 μL PI-PLC (0.45 mgtotal protein), 1 hour total reaction time. FIG. 12 illustrates a tablesummarizing data from this two-enzyme degumming system of the invention.

In another aspect, a PI-PLC enzyme of the invention can be used underthe same conditions described for PLC. These include chemical refiningof vegetable oils and water degumming of vegetable oils.

Example 4 Phospholipases with Improved Expression and Altered ProteaseResistance

The invention provides method for selectioning Phospholipase C variants(mutants) having improved expression in a glycosylating host and alteredresistance to secreted proteases.

Improved Expression in a Glycosylating Host.

Potential asparagines-linked glycosylation sites with the amino acidconsensus sequence, asparagine-any amino acid-serine or threonine (NXS/Tin the one letter amino acid code), were knocked out using mutagenesismethods to change the asparagines or the serine or the threonine in theglycosylation recognition motif to a different amino acid so thesequence no longer encodes a potential glycosylation site. Theelimination of the glycosylation sites was effected as indicated below:amino acid positions amino acid 63, amino acid 131, and amino acid 134,of the phospholipase C enzyme of the invention having an amino acidsequence as set forth in SEQ ID NO:2, encoded, e.g., by SEQ ID NO:1.This elimination of the glycosylation sites improved expression of thisvariant, active phospholipase C enzyme (PLC, SEQ ID NO:2) when theprotein was heterologously expressed in the yeast Pichia pastoris. Thisstrategy of reducing or eliminating potential glycosylation sites in thePLC enzyme can improve the expression of active PLC in any glycosylatinghost. Thus, the invention provides phospholipase enzymes (and thenucleic acids that encode them) having a sequence of any of theexemplary phospholipases of the invention with one or more or all of theglycosylation sites altered, as described above. Thus, the inventionprovides methods of making variant phospholipase coding sequences havingincreased expression in a host cell, where the method comprisesmodifying a phospholipase coding sequence of the invention such thatone, several or all N-linked glycosylation site coding motifs aremodified to a non-glycosylated motif. The invention also providesphospholipase coding sequence made by this process, and the enzymes theyencode.

Altered Resistance to Protease

The invention provides methods for making a variant phospholipase codingsequence encoding a phospholipase having increased resistance to aprotease comprising modifying an amino acid equivalent to position 131of SEQ ID NO:2 to one, several or all of the following residues: Lysine(K); Serine (S); Glycine (G); Arginine (R); Glutamine (Q); Alanine (A);Isoleucine (I); Histidine (H); Phenylalanine (F); Threonine (T);Methionine (M) Leucine (L), including variants to SEQ ID NO:2 (and thenucleic acid that encode them) having these exemplary modifications. Theinvention also provides isolated, synthetic or recombinantphospholipases encoded by a sequence made by this method. The inventionalso provides methods for making a variant phospholipase coding sequenceencoding a phospholipase having decreased resistance to a proteasecomprising modifying an amino acid equivalent to position 131 of SEQ IDNO:2 to one, several or all of the following residues: Tryptophan (W);Glutamate (E); Tyrosine (Y), including variants to SEQ ID NO:2 (and thenucleic acid that encode them) having these exemplary modifications. Theinvention also provides isolated, synthetic or recombinantphospholipases encoded by a sequence made by this method.

Supernatant containing a mixture of native secreted Pichia pastorisproteases is mixed and incubated with wild type and mutant PLC enzymepreparations. Reactions are quenched and degradation visualized bySDS-PAGE versus the no protease negative control. Degradation may alsodetermined by measurement of residual PLC activity. Novelty was derivedfrom the observation that certain mutations to knock-out glycosylationsignificantly change the susceptibility of the expressed phospholipaseto degradation during fermentation. An advantage to the method is directselection of mutants with increased or decreased resistance to theproteases secreted by the host organism during production.

This process of the invention can employ site directed mutagenesis(e.g., GSSM™) to change the amino acid sequence of a phospholipase Cenzyme of the invention, e.g., as shown below—a subsequence of SEQ IDNO:2 encoded by SEQ ID NO:1. Each of the amino acids highlighted in red(below) were changed from asparagine (N in single letter code) toAspartate (D), serine (S), or another amino acid as described below.These amino acids are designated as amino acid 63, amino acid 131, andamino acid 134 of the sequence below where tryptophan (W) is designatedamino acid 1. These mutations were made to increase the expression ofactive phospholipase C protein by reducing glycosylation of theexpressed protein in the Pichia pastoris expression system. These samemutations can increase expression of any active phospholipase C of theinvention in any other expression system that glycosylates asparagines(N-linked glycosylation) according to the NXS/T system where N isasparagine, X is any amino acid, and S/T is serine or threonine. Thus,the invention also provides a process for changing the susceptibility ofthe expressed phospholipase C by changing the amino acid in position131.

Amino acids 39-286 of SEQ ID NO:2:

NOTE: To count the positions changed, count the first amino acid (W) asposition 1.

The expressed phospholipase C variants were incubated in the presence ofP. pastoris proteases as described below and the following results wereobtained:

The following amino acids at amino acid position 131 of SEQ ID NO:2increased the resistance of the expressed phospholipase C to degradationby P. pastoris proteases: Lysine (K); Serine (S); Glycine (G); Arginine(R); Glutamine (Q); Alanine (A); Isoleucine (I); Histidine (H);Phenylalanine (F); Threonine (T); Methionine (M) Leucine (L). Thefollowing amino acids at amino acid position 131 of SEQ ID NO:2decreased the resistance of the expressed phospholipase C to degradationby P. pastoris proteases: Tryptophan (W); Glutamate (E); Tyro sine (Y).Thus, the invention provides variant phospholipases having any one of,or several or all of these modifications, depending on whether it wasdesired to increase or decrease the resistance of the expressedphospholipase C to degradation by protease. The invention providesvariant phospholipases having any one of, or several or all of thesemodifications in positions equivalent to position 131 of SEQ ID NO:2.Which residue is equivalent to position 131 of SEQ ID NO:2, and whetherany particular amino acid residue modification can increase or decreasethe resistance of the enzyme to degradation by a protease, can beroutinely and predictably ascertained by protocols well known in theart, e.g., the exemplary assay used to evaluate protease susceptibilityof the (SEQ ID NO:2, encoded by SEQ ID NO:1) phospholipase C describedbelow:

Buffers:

-   -   1.0 M MES, pH 6.2    -   0.7 M sodium acetate (“NaAc”), pH 5.2

Challenge:

-   -   Use separate 1.5 mL microfuge tubes    -   To 25 μL PLC enzyme sample add 5 μL NaAc or 7 μL MES buffer and        mix    -   Add 25 μL protease-containing Pichia pastoris supernatant and        mix    -   Add 2 μL 5% sodium azide and mix    -   Place tubes in floating rack in prewarmed beaker of water in a        humidified incubator    -   Controls include PLC+buffer+dH₂O and Pichia SN+buffer+dH₂O    -   Incubate from 0-24 hours, sampling multiple timepoints if        desired

Detection:

-   -   Visualize on SDS-PAGE by mixing samples 1:2 with sample buffer        containing 5 mM EDTA, heat 100° C., 4 minutes, cool, centrifuge,        mix, load 5 μL sample per lane, Coomassie stain.    -   Samples and timepoints may also be taken directly to standard        PLC activity assay.

Results: SDS-PAGE gels were run and the results are illustrated in FIG.17; which shows the results of the in vitro digestion experimentswherein the phospholipase C variants were incubated in crude proteaseextracts for up to 22 hr at 37° C. Each PLC mutant is named according tothe amino acid found in the “X” position of the sequence “DXD”(Aspartate at amino acid position 63-any amino acid at amino acidposition 131-Aspartate at amino acid position 134). The gels show thestability or sensitivity of the expressed PLC mutant protein followingincubation with crude protease. A stable mutant shows a PLC band ofsimilar staining intensity in the “−” (control no protease reaction) andthe “+” (reaction contains protease). A mutant more sensitive toprotease will show a reduction in PLC protein band staining intensity inthe “=” lane compared to the “−” lane.

Example 5 Process for Stable High Level Expression PLC

The invention provides a fermentation process for stable, high levelexpression and high specific activity of phospholipase enzymes, e.g.,PLC, in yeast cultures, e.g., Pichia pastoris cultures. The enzymesproduced by this method can be used, e.g., in vegetable oil refinement,such as soybean, canola, sunflower or other oils.

The invention provides a production process comprising characteristicsthat enable production of active phospholipase, e.g., PLC, in a yeastcell culture, e.g., Pichia pastoris, as fed-batch cultures at a g/lscale. Heterologous expression of active PLC protein in microbialcultures had occasionally been described in the literature only at themg/l scale. The process of the present invention is based, inter alia,on the finding that expression of PLC protein in Pichia cultures impairsthe MeOH uptake capacity, but no other studied physiological growthcharacteristics. In contrast to conventional heterologous proteinexpression in Pichia cultures, high co-feed rates (glucose/or glycerol)are required. In addition to improving enzyme productioncharacteristics, higher co-feeding also eliminates the expression ofgeneral protease activity which is correlated with PLC degradation. Inaddition, the poor MeOH utilization characteristics can be overcome,thereby improving the production characteristics further, by producingPLC in Pichia strains with a Mut⁺ phenotype without compromisingscalability challenges normally associated with a Mut⁺ phenotype (andare therefore, not used on industrial scale). Thus, this process of theinvention improves the production of active PLC by >50-fold (from 100U/ml using conventional methods to >5000 U/ml whole broth; >5 g/lprotein) compared to conditions that are normally applied in industrialscale Pichia systems. In addition, because PLC is a metallo-enzymerequiring binding of zinc for proper folding and activity, in one aspectthe invention comprises a zinc supplementation. This zincsupplementation strategy for the cultures of the invention renders thePLC activity nearly completely stable (<5% loss in activity) as a wholebroth, e.g., at 4° C. for >5 days. This significantly aides the recoveryprocess since 1) production of unstable protein activity continues toworsen during the recovery process, and 2) it allows for more processingflexibility, especially at large-scale.

Tryptophanyl Aminopeptidase Microplate Assay

The invention provides a Tryptophanyl Aminopeptidase Microplate Assay,which was developed for determination of relative tryptophanylaminopeptidase activities in Pichia fermentation timepoint samples. Thethroughput capacity of this assay is sufficient for sampling of multipletimepoints from numerous fermentations.

Materials and Methods

Buffer:

-   -   15 mM NaPO₄, 2 mM MnC12, pH 7.5, aq.    -   Substrate:    -   HTrp-AMC (Bachem, 11670)    -   Substrate solution:    -   Dissolve substrate to 10 mM in methanol    -   Add 100 μL 10 mM substrate to 6 mL of buffer

Samples:

-   -   Pichia fermentation timepoints    -   Centrifuge to remove cells.

Microplate preparation:

-   -   Aliquot 90 μl substrate solution per well of black 96-well for        each sample replicate, blanks and references    -   Place microplate on fluorescent microplate reader stage (e.g.        SpectraMax, Molecular Dynamics)

Sample addition and reaction kinetics:

-   -   Set-up fluorescent microplate reader:    -   Ex. 350 mn/Em. 460 nm; auto cutoff (455 μm); PMT medium; 3 reads        per well; autocalibrate “on”    -   RT    -   0-30 minute timecourse; read every 30 seconds    -   Initialize the instrument plate mix function to mix for 5        seconds before first read    -   Aliquot samples in a 96-well format and use a multichannel pipet        to transfer samples at 10 μL per well    -   With lid removed, replace microplate in microplate reader    -   Begin reading

Depending on the inherent activity of unknown samples, it may bedesirable to vary sample dilution, assay duration and kinetic sampling,all variables that can be determined by routine screening.

The substrate has been shown to be very stable under these conditionsand a negative control blank should show no increase in absorbance overtime.

Bodipy BSA Protease Microplate Assay

The invention provides a Bodipy BSA Protease Microplate Assay to aid inthe determination of general protease activity in Pichia fermentationtimepoint samples. The throughput capacity of this assay is sufficientfor sampling of multiple timepoints from numerous fermentations.

Materials and Methods

Substrate:

-   -   DQ BSA green (Molecular Probes, D12050)    -   Substrate solution:    -   Dissolve contents of one vial of substrate (1 mg) in 1 mL water        containing 0.1% sodium azide

Samples:

-   -   Pichia fermentation timepoints    -   Centrifuge to remove cells.

Positive control:

-   -   0.2 mg/mL subtilisin (Sigma, P5380) in 50 mM NaPO₄, pH 7.5    -   Serially dilute in water

Microplate preparation:

-   -   Aliquot 90 μl substrate solution per well of black 96-well for        each sample replicate, blanks and references

Sample addition and reaction:

-   -   Aliquot samples in a 96-well format and use a multichannel pipet        to transfer samples at 10 μL per well    -   Replace microplate cover, wrap with foil and place in humidified        incubator at 37° C. and allow to incubate 3-4 hours or overnight

Fluorescence measurement:

-   -   Set-up fluorescent microplate reader (SpectraMax):        -   Ex. 495 nm/Em. 525 nm; auto cutoff (515 nm); PMT low; 3            reads per well; autocalibrate “on”        -   RT

Bodipy BSA was selected as a general protease substrate. Lack ofhydrolysis of bodipy BSA does not indicate the absence of protease(s)but it has been shown to correlate to hydrolysis of PLC enzyme and lossof PLC activity. It has been demonstrated that BSA may be substitutedwith bodipy ovalbumin or casein.

In one aspect, it is useful to characterize protease activity across afermentation timecourse since the activity may be temporal andtransient.

The substrate has been shown to be very stable under these conditionsand a negative control blank should show no increase in absorbance overtime PLC activity measurement in whole culture broth or supernatant:

The invention provides a PLC activity measurement assay in whole culturebroth or supernatant; this is a modification of a method described,e.g., by Edward A. Dennis (1973) Kinetic dependence of phospholipase A2activity on the detergent Triton X-100. J. Lipid Res. 14:152-159, USP24/NF 19, Pancrealipase-Assay for lipase activity. Page 1256-1257. ThePLC activity measurement assay of the invention comprises:

Solutions:

-   -   100 mM Zinc Sulfate Solution    -   100 mM Calcium Chloride Solution    -   Substrate Solution (20 mM Phosphatidyl Choline, 40 mM Triton        X-100, 5 mM Calcium Chloride)    -   Dilution Buffer (0.1% Triton X-100, 1 mM Zinc Sulfate, 1% Gum        Arabic)

Assay Procedure:

-   -   Prepare dilutions of the samples to be assayed using the        dilution buffer (1.0% Gum Arabic, 1.0% Triton X-100, 1 mM zinc        sulfate). Prepare dilutions immediately before assay, using        ice-cold buffer, and store in an ice bath until used.    -   Transfer 20 mL of the substrate solution into a jacketed glass        vessel of about 50 mL capacity, the outer chamber of which is        connected to a thermostatically controlled water bath. Cover the        mixture, and stir continuously with a mechanical stirring        device. With mixture maintained at a temperature of 37±0.1° C.        pre-titrate the substrate with 0.01 NKOH VS, from a microburet        inserted through an opening in the cover, to adjust the pH to        7.3. Add 50 μL of enzyme dilution, and then continue        automatically to add the 0.01 N KOH VS for 6 minutes to maintain        the pH at 7.

In addition, standard PAGE gel electrophoresis, Western and Northernblot analysis on fermenter cultures as well as standard analysistechniques for on-line/off-line fermentation parameters (biomass levels,gas analysis etc.)

Generating the Mut⁺Phenotype Pichia Strains

The invention provides cells, cell systems and methods for expressingphospholipase C comprising using a Pichia strain with a Mut⁺ phenotype.The method comprises inserting a heterologous PLC-encoding nucleic acidin the Pichia strain. The cell is then cultured under conditions wherebythe PLC is expressed. The method can further comprise supplementing theculture conditions with zinc.

In one aspect, these methods, cells and cell systems use SEQ ID NO:2,which is a zinc-requiring metalloenzyme. In one aspect, it is used at 3moles/mole. It has a MW of approximately 28 kDa and a pI ofapproximately 5.2, and has a broad substrate tolerance: PC>PE>PS>>PI.The unprocessed enzyme has a signal sequence of 24 amino acids, aprosequence of 13 amino acids, and a “mature” enzyme of 245 amino acidresidues.

In one aspect, the Mut⁺ Pichia strains of the invention have two copiesof alcohol oxidase (AOX) genes, AOX1 and AOX2, affected duringtransformation (“Mut” stands for “Methanol Utilization”), as follows:

Mut⁺

-   -   Single crossover event, AOX1 and AOX2 genes intact    -   Growth and expression on methanol alone. Co-feeding possible

Mut^(S)

-   -   Double crossover event disrupts the AOX1 gene    -   Growth and expression improved with co-feeding

Mut⁻

-   -   Recombination event disrupts AOX1 and 2 genes    -   Cannot metabolize methanol, requires co-feeding        In summary: Mut⁻<Mut^(s) _(plc)<Mut^(s)/Mut⁺ _(plc)<Mut⁺

There are fermentation differences between Mut⁺ and Mut^(s), including:

-   -   Optimal Induction Concentration of Methanol    -   Oxygen Consumption Rate    -   Mut⁺ grows faster than Mut^(s) on Methanol due to faster uptake        capacity    -   Ease of Transition Period after Induction    -   Mut⁺ not used for expression at large scale        -   Aeration/cooling capacity, MeOH sensitivity

The methanol utilization pathway in Pichia pastoris is well known in theart. Alcohol oxidase (AOX) catalyzes the conversion of methanol toformaldehyde; thus, if the AOX is overexpressed, results in a “pickled”yeast cell.

An exemplary fermentation protocol for Pichia pastoris used in oneaspect of the invention comprises:

Seed Culture (flask or tank)

-   -   Batch fermentation in rich medium to enhance biomass

Fed-Batch Fermentor Culture

-   -   Batch Phase (Glycerol)        -   Biomass growth as initial carbon source is consumed.    -   Glucose or Glycerol Feeding Phase        -   Addition of feed triggered by D.O. content or            linear/exponential feeding        -   Growth to sufficient biomass for induction and expression            (absence of Ethanol, C-limited)    -   Methanol Induction        -   Addition of feed regulated (D.O. %, MeOH sensor, RQ) or            preset feeding profiles        -   Co-feeding with glucose or glycerol dependent on phenotype            and expression parameters        -   Mut⁺ Induction at 1-3 g/L MeOH        -   Mut^(s) Induction at 4-7 g/L MeOH

FIG. 18 illustrates the results of a batch fermentor culture, asdiscussed above, using only glycerol. Protease activity is from anendogenous protease in Pichia. The batch fermentation can be rich mediumto enhance biomass. As noted in FIG. 18, the progressive increase inprotease activity beginning at about 69 hours corresponds to aprogressive decrease in PLC activity. A higher co-feed rate of glycerol(glyc) improves active PLC expression and decreases (eliminates)protease production, as the following data summary table illustrates:

C-source Co-feed before/ Induc- PLC MeOH rate after tion activityconsumed Bodipy Final (ml/min) induction OD (U/ml sup) (L) protease OD0.5 Glyc/Glyc 250-300 100 1 Yes 450 1.5 Glyc/Glyc 1100 1.7 No 680 2Glyc/Glyc 1550 1.3 No 860 2.5 Glyc/Glyc 1550 1.4 No 900 3 Glyc/Glyc 17151.4 No 820

These studies were done in 30-L BB fermenters with DSD-PLC. The OUR, orVol. Oxygen Uptake Rate (“OUR”), as an ‘overall culture health’indicator or ‘Biomarker’ for good expression, was measured. FIG. 19illustrates the results of such a study, an OUR profile comparison ofcultures of P. pastoris MutS 30 L cultures producing DSD-PLC, using 1700U/ml, 1100 U/ml and 100 U/ml PLC, 30° C., glycerol co-feed, as discussedabove.

FIG. 20 illustrates a methanol consumption profile comparison in P.pastoris MutS 30 L cultures producing DSD-PLC, pH 6.2 (1100 U/ml and 100U/ml PLC), or a heterologous protein, with a glycerol co-feed, asdiscussed above. This was a demand-driven MeOH feeding, and the residualMeOH level was controlled at 4 g/l.

In addition, Mut⁺ phenotype improves active PLC expression and enhancesMeOH uptake, as this data table summarizes:

Co-feed Induc- PLC MeOH rate tion activity consumed Bodipy Final Mut(ml/min) OD (U/ml sup) (L) protease OD S 0.5 250-300 100 1 Yes 450 1.51100 1.7 No 680 2 1550 1.3 No 860 2.5 1550 1.4 No 900 3 1715 1.4 No820 + 0.5 250-300 1001 5.6 yes 871 0.5 1200 7 No 908 1 1786 5.9 No 988 12010 6.8 No 930 1 1768 7.9 No 700 1.5 2669 10 No 701 1.5 2693 7.1 No 8181.5 2597 8.1 No 804 2 2154 8.3 No 752

PLC does not seem to affect physiological growth characteristics of thisMut⁺ phenotype strain—which expresses recombinant PLC SEQ ID NO:2, in a6× copy number, the data illustrated in FIG. 21, an OUR profile as setforth in the figure description. This is a supply-driven MeOH feedingwith no residual glucose or MeOH in Mut⁺ cultures.

Additionally, the quality of PLC protein produced is unpredictablyvariable, e.g., <<or >>50% of total PLC protein is active, asillustrated by the representation of the results from SDS-PAGE, in FIG.22. The OUR profile (discussed above) graphic summary of data isinserted into the upper section of the SDS-PAGE illustration. Thecontrol is designated JG=0.5 μl 1.6 mg ml-1. There was no correlationwith protease or aminopeptidase activity. A significant quantity ofactive PLC was located intracellularly, as illustrated in FIG. 23 (alsoshowing the study's protocol), where >700 U/ml PLC was detectedintracellularly (in FIG. 23, PLC (SEQ ID NO:2)+ an alpha signal peptide(from Saccharomyces)+glycosylation). Morphological changes werecorrelated with active PLC concentration, as illustrated in FIG. 24.Magnitude of the morphological change was strain and C-source dependent.

Increased Zn did not boost expression in a Pichia strain having 2× copynumber Mut+ SEQ ID NO:2 with DSD mutation, as summarizes in the datachart, below (excess over 1× supplied via co-feed) (first, upper row isempty vector control). Increased Zn did improve storage stability aswhole broth (similar activity level after >100 h at 4° C.) and overallrobustness of process.

MeOH Base 70% (v/v) PLC Zn (L) (L) Glycerol (L) OD600 (U/ml) 1X 7.1 2.39.6 765 0 (2.2 mM) 0.2X 7.4 2.1 8.6 731 392 1X 7.1 2.8 9.0 776 2700 4X6.1 2.2 10 780 2448 12X 6.4 2.3 9.8 776 2498

FIG. 25 graphically summarizes data showing the status of a PLCproduction performance at 95 h TFT (total fermentation time) in Pichia.The five bars on the right side of the graph show results from the “Zeostrain”, or Zeocin adaptation of the PLC producing Pichia pastorisstrain. This strain is an antibiotic-resistant markerless strainexpressing as a heterologous gene a PLC of the invention (SEQ ID NO:2)in a Pichia pastoris strain. It has been demonstrated that by adaptingthe strain with zeocin, an antibiotic, one can obtain a new stablestrain with greatly improved expression level for the protein ofinterest.

The original antibiotic-resistant markerless strain, strain #1(containing SEQ ID NO:2), was grown in a series of dilution steps, eachtime with an increasing concentration of zeocin, which is an antibiotic.On each step, a portion of the culture from previous step was diluted toan optical density at 600 nm (OD600) of 1.0 with fresh medium and anincreasing amount of zeocin was added to the new culture for another 24hours of growth. At the final stage, a zeocin concentration of 200 ug/mlwas used and the final culture was streaked to a MD/YPD plate to allowindividual colonies to grow. It was found that the colonies from thefinal stage culture show high tolerance to zeocin, while the parentstrain exhibits very little tolerance. One of the colonies, strain #2(containing SEQ ID NO:2), showed dramatic improvement (about 70% higher)in PLC expression compared to the original PLC strain, strain #1. It wasalso demonstrated that strain #2 is stable both in zeocin tolerance andPLC expression after a 40-generation passage, indicating that the newstrain acquired the “permanent” trait of high PLC expression and zeocintolerance.

A high level of PLC activity was achieved using the “Zeo strain” (ZeocinPichia adaptation) of the invention: 4100 u/ml achieved in mini-tanks.This result comes from the Pichia strain comprising 6×DSD SEQ ID NO:2.Briefly, this SEQ ID NO:2-expressing strain was “adapted” by growing itin a series of steps, each with increasing concentration of zeocin.Apparently, this adaptation process forced some changes (in molecular orgenetic level) to the strain/construct and resulted in significantimprovement of PLC activity level. Exemplary results are:

-   -   Tank 1, 2, and 4 (each representing different colonies) all        out-performed the original pre-adapted SEQ ID NO:2-expressing        strain, with tank 1 & 4 both got to 4100 u/ml and tank 2 got to        3500 u/ml.    -   Tank 1 & 4 got to over 3000 u/ml as early as in 75 hrs,        representing a much faster activity accumulation comparing to        the original pre-adapted SEQ ID NO:2-expressing strain (which is        normally well below 2000 u/ml at the time).

Details of the Experimental Design and Result are:

Rationale of Zeocin Adaptation:

Earlier stage of work on PLC expression was done in the Pichia pPICZavector, which contains the zeocin-resistance marker. Zeocin was thusused for transformation selection. Later on, we switched to the AMR-lessversion construct to develop commercial product candidates. While doingmini-tank fermentations, we observed a significant drop of PLC activitylevel obtained using the AMR-less constructs: supernatant activityreached 4000 u/ml in pPICZa-DSD constructs, whereas only ca. 2000 u/mlwas obtained in the 2×DSD. Significant physiological differences, e.g.,lower methanol consumption rates and a lot more cell lysis, were alsoobserved with the AMR-less constructs, especially when testing highercopy number (5×, 6×) constructs using the same fermentation protocols.

With one of the apparent differences between the pPIZa construct and theAMR-less construct being the use of zeocin in transformation, thequestion was raised on what the cells might have gone through withzeocin selection. The invention provides growing the AMR-less constructin the presence of zeocin—the cells then go through some changesbeneficial to PLC expression.

Zeocin Adaptation Experiment on 2×DSD:

The experiment was first used with the 2×DSD (as it was the transfermolecule at the time). The study started with a zeocin concentration of1 ug/ml (“zeo 1”) and grew the culture for 24 hrs. From there, stepincrease of zeocin concentration to zeo 5, zeo 10, zeo 15, zeo 20, zeo40, zeo 60, zeo 80, zeo 100 and finally to zeo 200 were carried out (zeo100 is normally used for transformation selection). Each step freshmedium was used and previous stage culture was used to inoculate thenext stage culture with OD of 1.0 and grown for 24 hrs. Cultures of eachstage were also streaked to YPD plates for preservation and to obtainindividual colonies.

Mini-Tank Fermentation Results of Zeo-Adapted Colonies:

To test the effects of zeocin adaptation, a dozen of colonies from zeo200 and zeo 100 cultures (that were streaked to YPD plates) was pickedand screened with mini-tanks. The results are summarized in slide 6. Wewere able to find several colonies that significantly outperformed theoriginal construct (Pichia strain comprising SEQ ID NO:2). Among them,colony #5 from zeo 200 culture showed about 50% improvement on PLCactivity level. Observations on the screening:

-   -   There were no apparent differences on growth profiles between        the zeo-adapted cultures and the original SEQ ID NO:2-expressing        strain.    -   Although stability of the adapted cultures was not extensively        tested, they were re-streaked several times on YPD and/or MD        plates without the presence of zeocin. All fermentation was also        done without the presence of zeocin.    -   There were apparent colony to colony variations, both on growth        and on PLC expression.    -   Some technical problems with the fermentation might be partly        responsible for the variations.

Zeocin Adaptation Experiment on 6×DSD:

Encouraged by the results from the zeo-adapted 2×DSD, we then carriedthe same experiment on the 6×DSD (which at the time was determined asbeing superior to the 2×DSD). We started with zeocin concentration of 5ug/ml (“zeo 5”) and grew the culture for ˜24 hrs. From there, stepincrease of zeocin concentration to zeo 15, zeo 30, zeo 50, zeo 100 andfinally to zeo 200 were carried out. Same as with the 2×DSD, each stepfresh medium was used and previous stage culture was used to inoculatethe next stage culture with OD of 1.0 and grown for 24 hrs. Cultures ofeach stage were also streaked to YPD plates for preservation and toobtain individual colonies.

Mini-Tank Results of Zeo-Adapted 6×DSD Colonies:

Six colonies from the zeo 200 culture (that was streaked to MD plate)were picked and tested together with the original SEQ ID NO:2-expressingstrain in the mini-tanks. Key observations are as below:

-   -   All three colonies (tank 1, 2, and 4) out-performed the original        SEQ ID NO:2-expressing strain, with tank 1 & 4 both got to 4100        u/ml and tank 2 got to 3500 u/ml.    -   Tank 1 & 4 got to over 3000 u/ml as early as in 75 hrs,        representing a much faster activity accumulation comparing to        the SEQ ID NO:2-expressing strain (which is normally well below        2000 u/ml at the time).    -   PLC protein level also seems to be higher in tanks 1, 2, & 4        comparing to the 3000 u/ml run in 10-L tank (see slide 4). It is        thus not clear whether apparent specific activity is higher in        tanks 1, 2, & 4, i.e., whether the PLC being produced is        different than from the original SEQ ID NO:2-expressing strain.        The control, tank 7 & 8, did not get to 3000 u/ml this time.        It's not clear whether tank 1, 2, & 4 might be able to reach        even higher level. Note that the percent increase (35%, 4100        u/ml vs 3000 u/ml) is smaller than the 2× adapted culture.    -   A summary of expression screening from the 6×DSD zeocin-adapted        colonies is found in FIG. 26. The highest activity level seen        with the original strain was ˜3000 u/ml (mini-tank & 10-L); the        level achieved with zeocin-adapted 6×DSD was 4100 u/ml (˜35%        increase). FIG. 27 illustrates data showing that PLC protein        level was higher in tanks 1, 2, & 4 comparing to the 3000 u/ml        run in 10-L tank (and tank conditions), as discussed above (the        gel loading was at 1.0 ul of 5× diluted broth, 0.2 ul of whole        broth). FIG. 28 shows the growth comparison of zeo-adapted        colonies vs control. The Zeocin-adapted 6×DSD colonies have        similar growth profile compared to the original SEQ ID        NO:2-expressing strain (6×DSD).

The Qp of secreted protein in C-limited aerobic yeast cultures isgenerally 0.5-2.5 mg/g.h-1 at μ=0.10 h⁻¹. Based on protein content of400 mg/g DW, ‘metabolic burden’ is <10% of overall protein productionrate. PLC mRNA level remains high throughout fermentation and does notcorrelate with expression. Based on 5 g/l (150 g) PLC protein, less than0.1 mol C/h of total 5 mol C/h (2% of total C consumed) goes to PLCcarbon and ˜25% goes to biomass. PLC activity does not seem to impactgeneral growth physiological characteristics under these productionconditions (except MeOH utilization capacity is affected).

In summary, the invention provides zeocin-resistant yeast cell systems,such as yeast cells, cell lines and/or individual cells, for expressinga heterologous protein (e.g., an enzyme, such as a PLC) made by aprocess comprising the steps of providing a Pichia sp. (e.g., P.pastoris) cell comprising a heterologous nucleic acid (e.g., a vectorcomprising an enzyme coding sequence; an ORF operably linked to apromoter) capable of expressing a heterologous protein; culturing thecell(s) under conditions comprising zeocin at an initial concentration(a concentration low enough that some cells survive, but, high enough toselect for antibiotic resistant cells); selecting cells resistant to theinitial concentration of zeocin, and reculturing under conditionscomprising a higher concentration of zeocin; and selecting the cellsresistant to the higher concentration of zeocin. The invention alsoprovides yeast cells, cell lines and/or individual cells made by thisprocess. Routine screening can determine which initial concentration ofantibiotic to use, how many rounds of selection are needed, or desired,and how quickly to increase concentrations of antibiotic betweenselection rounds.

Example 6 Thermostable PLC

The invention provide thermostable phospholipase enzymes. Thethermostability for the exemplary enzyme having a sequence as set forthin SEQ ID NO:2 was demonstrated. Thermostability of comparablephospholipids of the invention was demonstrated using SEQ ID NO:2. Theactivity of SEQ ID NO:2 was tested in two different systems: aqueous andin oil. In the aqueous system, a surrogate substrate (p-nppc) was usedto measure activity; the enzyme began to loose activity at 86 C. Howeverin the oil assays, the enzyme showed good activity in hydrolyzing PC andPE substrates present in soy oil at 85 C. Tm of the same enzyme waschecked and found that it was 86 C @15 mg/mL, and not reversible.

FIG. 29 illustrates the results of an 85° C. heating experiment with 10U of SEQ ID NO:2, with the conditions indicated in the figure. FIG. 30illustrates NMR data summarizing this heating experiment. FIGS. 31, 32and 33 illustrate data summarizing the thermal stability of SEQ ID NO:2using p-NPPC, at the conditions shown in the figure. FIG. 34 illustratesdata from DSC analysis showing the thermostability of SEQ ID NO:2, withthe enzyme at a concentration of 15 mg/mL and the Tm at 86° C.

1: An isolated, synthetic or recombinant nucleic acid comprising (i) anucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more or complete sequence identity to (a) a nucleic acidthat encodes the polypeptide having a sequence as set forth in SEQ IDNO:2; (b) a nucleic acid that encodes the polypeptide having a sequenceas set forth in SEQ ID NO:2, but lacking a signal sequence; or, to (c)SEQ ID NO:1, wherein the nucleic acid encodes at least one polypeptidehaving a phospholipase activity; (ii) a sequence that hybridizes understringent conditions to SEQ ID NO:1, or to a nucleic acid sequence thatencodes the polypeptide having a sequence as set forth in SEQ ID NO:2,wherein the stringent conditions include a wash step comprising a washin 0.2×SSC at a temperature of about 65° C. for about 15 minutes,wherein the nucleic acid encodes at least one polypeptide having aphospholipase activity; (iii) the nucleic acid of (i), wherein thesequence identities are determined by analysis with a sequencecomparison algorithm comprising a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall-p blastp-d “nr pataa”−F F, and allother options are set to default; (iv) the nucleic acid of any of (i) to(iii), wherein the nucleic acid encodes: (A) SEQ ID NO:175, which isequivalent to SEQ ID NO:2 minus a leader sequence, and having three (3)amino acid residue changes as compared to SEQ ID NO:2, or, (B) SEQ IDNO:176, which is equivalent to SEQ ID NO:2 but having three (3) aminoacid residue changes as compared to SEQ ID NO:2; (v) the nucleic acid ofany of (i) to (iv), wherein the phospholipase activity comprises:catalyzing hydrolysis of a glycerolphosphate ester linkage; catalyzinghydrolysis of an ester linkage in a phospholipid in a vegetable oil oran oilseed phospholipid; a phospholipase C(PLC) activity; aphospholipase A (PLA) activity; a phospholipase B (PLB) activity; aphospholipase D (PLD) activity; a phospholipase D1 or a phospholipase D2activity; hydrolysis of a glycoprotein; hydrolysis of a glycoprotein ina potato tuber; a patatin enzymatic activity; or, a lipid acyl hydrolase(LAH) activity; (vi) the nucleic acid of any of (i) to (v), wherein thephospholipase activity comprises a combination of one or more ofphospholipase activities, or the phospholipase activity comprises PLCand PLA activity; PLB and PLA activity; PLC and PLD activity; PLC andPLB activity; PLB and patatin activity; PLC and patatin activity; PLDand PLA; PLD, PLA, PLB and PLC activity; or PLD, PLA, PLB, PLC andpatatin activity, or the phospholipase activity compriseslysophospholipase (LPL) activity or lysophospholipase-transacylase(LPTA) activity or lysophospholipase (LPL) activity andlysophospholipase-transacylase (LPTA) activity, (vii) the nucleic acidof any of (i) to (vi), wherein the phospholipase activity comprisescatalyzing hydrolysis of a glycerolphosphate ester linkage inphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), and/or phosphatidicacid or a combination thereof, (viii) the nucleic acid of any of (i) to(vii), wherein the phospholipase activity is thermostable, or thepolypeptide retains a phospholipase activity under conditions comprisinga temperature range of between about 37° C. to about 95° C., or betweenabout 55° C. to about 85° C., or between about 70° C. to about 75° C.,or between about 70° C. to about 95° C., or between about 90° C. toabout 95° C.; (ix) the nucleic acid of any of (i) to (viii), wherein thephospholipase activity is thermotolerant, or the polypeptide retains aphospholipase activity after exposure to a temperature in the range fromgreater than 37° C. to about 95° C., from greater than 55° C. to about85° C., or between about 70° C. to about 75° C., or from greater than90° C. to about 95° C.; (x) the nucleic acid of any of (i) to (ix),wherein the nucleic acid further comprises a heterologous nucleic acidsequence; (xi) the nucleic acid of (x), wherein the heterologous nucleicacid sequence encodes a heterologous polypeptide; (xii) the nucleic acidof (xi), wherein the heterologous polypeptide comprises or consists of asignal sequence (a signal peptide), a catalytic domain, a linkersequence, a protease cleavage recognition site, a non-phospholipaseenzyme sequence, a phospholipase enzyme sequence; or (xiii) a nucleicacid comprising a sequence completely (fully) complementary to thenucleic acid sequence of any of (i) to (xii). 2-28. (canceled) 29: Aprobe for identifying or isolating a nucleic acid encoding a polypeptidewith a phospholipase activity, wherein the probe comprises (A) (a) anucleic acid sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:2, SEQ ID NO:175 or SEQ ID NO:176, or (b) SEQ IDNO:1, or (c) the nucleic acid of claim 1, wherein the probe identifiesthe nucleic acid by binding or hybridization, or (B) the probe of (A),wherein the probe comprises a detectable agent, a radioactive isotope, afluorescent dye or an enzyme capable of catalyzing the formation of adetectable product. 30-40. (canceled) 41: An expression cassettecomprising a nucleic acid comprising the sequence of claim
 1. 42: Avector or a cloning vehicle comprising (a) a nucleic acid comprising thesequence of claim 1, or (b) the vector or cloning vehicle of (a),wherein the cloning vehicle comprises a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome, or the viral vector comprises an adenovirus vector, aretroviral vector or an adeno-associated viral vector, or the vector ora cloning vehicle comprises or is contained within a bacterialartificial chromosome (BAC), a plasmid, a bacteriophage P1-derivedvector (PAC), a yeast artificial chromosome (YAC), or a mammalianartificial chromosome (MAC). 43-45. (canceled) 46: A transformed cellcomprising (a) a nucleic acid comprising the sequence of claim 1, or theexpression cassette of claim 41, or the vector or cloning vehicle ofclaim 42, or (b) the transformed cell of (a), wherein the cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insectcell or a plant cell. 47-48. (canceled) 49: A transgenic non-humananimal comprising (a) the sequence of claim 1, or (b) the transgenicnon-human animal of (a), wherein the animal is a mouse, a goat, arabbit, a sheep, a pig or a cow.
 50. (canceled) 51: A transgenic plantor seed comprising (a) the sequence of claim 1, or (b) the transgenicplant or seed of (a), wherein the plant is a corn plant, a sorghumplant, a potato plant, a tomato plant, a wheat plant, an oilseed plant,a rapeseed plant, a soybean plant, a rice plant, a barley plant, agrass, a cottonseed, a palm, a sesame plant, a peanut plant, a sunflowerplant or a tobacco plant, or (c) the transgenic plant or seed of (a),wherein the seed is a corn seed, a wheat kernel, an oilseed, a rapeseed,a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice,a barley, a peanut, a cottonseed, a palm, a peanut, a sesame seed, asunflower seed or a tobacco plant seed. 52-54. (canceled) 55: Anantisense oligonucleotide comprising (a) a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions tothe sequence of claim 1, or (b) the antisense oligonucleotide of (a),wherein the antisense oligonucleotide is between about 10 to 50, about20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases inlength.
 56. (canceled) 57: A method of inhibiting the translation of aphospholipase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to the sequence of claim
 1. 58: A double-stranded inhibitoryRNA (RNAi) molecule comprising (a) a duplex nucleotide version of thesequence of claim 1, or (b) the double-stranded inhibitory RNA (RNAi) of(a), wherein the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 or more duplex nucleotides in length.
 59. (canceled) 60: A method ofinhibiting the expression of a phospholipase in a cell comprisingadministering to the cell or expressing in the cell a double-strandedinhibitory RNA (iRNA), wherein the RNA comprises the double-strandedinhibitory RNA (RNAi) molecule sequence of claim
 58. 61: An isolated,synthetic or recombinant polypeptide having phospholipase activity orcapable of acting as an immunogen to generate an antibody specific forwherein the polypeptide comprises: (i) an amino acid sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more orcomplete sequence identity to the amino acid sequence of SEQ ID NO:2;(ii) an amino acid sequence encoded by the nucleic acid of any one ofclaim 1 (i) to claim 1 (xi); (iii) the amino acid sequence of SEQ IDNO:2 and having at least one amino acid modification selected from thegroup consisting of N63D, N131S and N134D, or having at least two of theamino acid modifications selected from the group consisting of N63D,N131S and N134; (iv) comprising the amino acid sequence of (i), (ii) or(iii) and lacking a homologous signal sequence; (v) comprising the aminoacid sequence of (i), (ii), (iii) or (iv) and further comprising aheterologous amino acid sequence; (vi) comprising the sequence of (v),wherein the heterologous amino acid sequence comprises or consists of asignal sequence (a signal peptide), a catalytic domain, a linkersequence, a protease cleavage recognition site, a non-phospholipaseenzyme sequence, a phospholipase enzyme sequence; (vii) comprising theamino acid sequence of (v) wherein the heterologous sequence amino acidsequence comprises an N-terminal identification peptide or a peptide orprotein that increases stability or simplifies purification of thepolypeptide, (viii) the amino acid sequence of (i), wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm comprising a BLAST version 2.2.2 algorithm where a filteringsetting is set to blastall-p blastp-d “nr pataa”-F F, and all otheroptions are set to default; (ix) the amino acid sequence of any of (i)to (viii), wherein the polypeptide comprises a sequence as set forth in:(A) SEQ ID NO:175, which is equivalent to SEQ ID NO:2 minus a leadersequence, and having three (3) amino acid residue changes as compared toSEQ ID NO:2, or, (B) SEQ ID NO:176, which is equivalent to SEQ ID NO:2but having three (3) amino acid residue changes as compared to SEQ IDNO:2; (x) the amino acid sequence of any of (i) to (ix), wherein thephospholipase activity comprises: catalyzing hydrolysis of aglycerolphosphate ester linkage; catalyzing hydrolysis of an esterlinkage in a phospholipid in a vegetable oil or an oilseed phospholipid;a phospholipase C (PLC) activity; a phospholipase A (PLA) activity; aphospholipase B (PLB) activity; a phospholipase D (PLD) activity; aphospholipase D1 or a phospholipase D2 activity; hydrolysis of aglycoprotein; hydrolysis of a glycoprotein in a potato tuber; a patatinenzymatic activity; or, a lipid acyl hydrolase (LAH) activity; (xi) theamino acid sequence of any of (i) to (x), wherein the phospholipaseactivity comprises a combination of one or more of phospholipaseactivities, or the phospholipase activity comprises PLC and PLAactivity; PLB and PLA activity; PLC and PLD activity; PLC and PLBactivity; PLB and patatin activity; PLC and patatin activity; PLD andPLA; PLD, PLA, PLB and PLC activity; or PLD, PLA, PLB, PLC and patatinactivity, or the phospholipase activity comprises lysophospholipase(LPL) activity or lysophospholipase-transacylase (LPTA) activity orlysophospholipase (LPL) activity and lysophospholipase-transacylase(LPTA) activity, (xii) the amino acid sequence of any of (i) to (xi),wherein the phospholipase activity comprises catalyzing hydrolysis of aglycerolphosphate ester linkage in phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylinositol (PI), and/or phosphatidic acid or a combinationthereof, (xiii) the amino acid sequence of any of (i) to (xii), whereinthe phospholipase activity is thermostable, or the polypeptide retains aphospholipase activity under conditions comprising a temperature rangeof between about 37° C. to about 95° C., or between about 55° C. toabout 85° C., or between about 70° C. to about 75° C., or between about70° C. to about 95° C., or between about 90° C. to about 95° C.; (xiv)the amino acid sequence of any of (i) to (xiii), wherein thephospholipase activity is thermotolerant, or the polypeptide retains aphospholipase activity after exposure to a temperature in the range fromgreater than 37° C. to about 95° C., from greater than 55° C. to about85° C., or between about 70° C. to about 75° C., or from greater than90° C. to about 95° C.; (xv) the amino acid sequence of any of (i) to(xiv), wherein the phospholipase activity comprises a specific activityat about 37° C. in the range from about 10 to about 100 units permilligram, from about 100 to about 1000 units per milligram of protein,from about 500 to about 750 units per milligram of protein, from about500 to about 1200 units per milligram of protein, or from about 750 toabout 1000 units per milligram of protein; (xvi) the amino acid sequenceof any of (i) to (xiv), wherein the thermotolerance comprises retentionof at least half of the specific activity of the phospholipase at 37° C.after being heated to an elevated temperature; (xvii) the amino acidsequence of any of (i) to (xvi), wherein the thermotolerance comprisesretention of specific activity at 37° C. in the range from about 500 toabout 1200 units per milligram of protein after being heated to anelevated temperature; (xviii) the amino acid sequence of any of (i) to(xvi), wherein the polypeptide comprises at least one glycosylationsite, and optionally the glycosylation is an N-linked glycosylation, orthe polypeptide is glycosylated after being expressed in an P. pastorisor an S. pombe; (xix) the amino acid sequence of any of (i) to (xix),wherein the polypeptide retains a phospholipase activity underconditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0,or more acidic; (xx) the amino acid sequence of any of (i) to (xix),wherein the polypeptide retains a phospholipase activity underconditions comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10or pH 10.5, or more basic; or (xxi) the amino acid sequence of any of(i) to (xx), wherein the polypeptide comprises at least one modificationcomprising or consisting of an acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof a phosphatidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of an aminoacid or arginylation. 62-92. (canceled) 93: A protein preparationcomprising the polypeptide of claim 61, wherein the protein preparationcomprises a buffer, a liquid, a solid or a gel. 94: A heterodimercomprising (a) the polypeptide of claim 61 and a second domain, or (b)the heterodimer of (a), wherein the second domain is a polypeptide andthe heterodimer is a fusion protein, or the second domain comprises anepitope or a tag. 95-96. (canceled) 97: A homodimer comprising thepolypeptide of claim
 61. 98: An immobilized polypeptide, wherein thepolypeptide comprises (a) the polypeptide of claim 61; or (b) theimmobilized polypeptide of (a), wherein the polypeptide is immobilizedon a cell, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, an arrayor a capillary tube.
 99. (canceled) 100: An array comprising theimmobilized polypeptide of claim 61, or an immobilized nucleic acid,wherein the nucleic acid comprises the sequence of claim 1, or acombination thereof.
 101. (canceled) 102: An isolated, synthetic orrecombinant antibody that specifically binds to the polypeptide of claim61, and optionally the antibody is a monoclonal or a polyclonalantibody.
 103. (canceled) 104: A hybridoma comprising an antibody thatspecifically binds to the polypeptide of claim
 61. 105: A method ofisolating or identifying a polypeptide with a phospholipase activitycomprising the steps of: (a) providing the antibody of claim 102; (b)providing a sample comprising polypeptides; and (c) contacting thesample of step (b) with the antibody of step (a) under conditionswherein the antibody can specifically bind to the polypeptide, therebyisolating or identifying a polypeptide having a phospholipase activity.106: A method of making an anti-phospholipase antibody comprising (a)administering to a non-human animal the nucleic acid of claim 1 in anamount sufficient to generate a humoral immune response, thereby makingan anti-phospholipase antibody, or, (b) administering to a non-humananimal the polypeptide of claim 61 in an amount sufficient to generate ahumoral immune response, thereby making an anti-phospholipase antibody.107. (canceled) 108: A method of producing a recombinant polypeptidecomprising: (A) (a) providing a nucleic acid operably linked to apromoter, wherein the nucleic acid comprises the sequence of claim 1;and (b) expressing the nucleic acid of step (a) under conditions thatallow expression of the polypeptide, thereby producing a recombinantpolypeptide, or (B) the method of (A), wherein the method furthercomprises transforming a host cell with the nucleic acid of step (a)followed by expressing the nucleic acid of step (a), thereby producing arecombinant polypeptide in a transformed cell.
 109. (canceled) 110: Amethod for identifying a polypeptide having a phospholipase activitycomprising: (a) providing the polypeptide of claim 65; (b) providing aphospholipase substrate; and (c) contacting the polypeptide with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having a phospholipaseactivity. 111: A method for identifying a phospholipase substratecomprising: (a) providing the polypeptide of claim 65; (b) providing atest substrate; and (c) contacting the polypeptide of step (a) with thetest substrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofa reaction product identifies the test substrate as a phospholipasesubstrate; or, 112: A method of determining whether a test compoundspecifically binds to a polypeptide comprising: (i) (a) expressing anucleic acid or a vector comprising the nucleic acid under conditionspermissive for translation of the nucleic acid to a polypeptide, whereinthe nucleic acid has the sequence of claim 1; (b) providing a testcompound; (c) contacting the polypeptide with the test compound; and (d)determining whether the test compound of step (b) specifically binds tothe polypeptide; or, (ii) (a) providing the polypeptide of claim 61; (b)providing a test compound; (c) contacting the polypeptide with the testcompound; and (d) determining whether the test compound of step (b)specifically binds to the polypeptide.
 113. (canceled) 114: A method foridentifying a modulator of a phospholipase activity comprising: (A) (a)providing the polypeptide of claim 61; (b) providing a test compound;(c) contacting the polypeptide of step (a) with the test compound ofstep (b) and measuring an activity of the phospholipase, wherein achange in the phospholipase activity measured in the presence of thetest compound compared to the activity in the absence of the testcompound provides a determination that the test compound modulates thephospholipase activity, or (B) the method of (A), wherein thephospholipase activity is measured by providing a phospholipasesubstrate and detecting a decrease in the amount of the substrate or anincrease in the amount of a reaction product, or, an increase in theamount of the substrate or a decrease in the amount of a reactionproduct, or (C) the method of (B), wherein a decrease in the amount ofthe substrate or an increase in the amount of the reaction product withthe test compound as compared to the amount of substrate or reactionproduct without the test compound identifies the test compound as anactivator of phospholipase activity, or (D) the method of (B), whereinan increase in the amount of the substrate or a decrease in the amountof the reaction product with the test compound as compared to the amountof substrate or reaction product without the test compound identifiesthe test compound as an inhibitor of phospholipase activity. 115-117.(canceled) 118: A computer system comprising (A) a processor and a datastorage device wherein said data storage device has stored thereon apolypeptide sequence or a nucleic acid sequence, wherein the polypeptidesequence comprises the sequence of claim 61, a polypeptide encoded bythe nucleic acid of claim 1, or (B) the computer system of (A), whereinthe computer system further comprises a sequence comparison algorithmand a data storage device having at least one reference sequence storedthereon, or (C) the computer system of (B), wherein the sequencecomparison algorithm comprises a computer program that indicatespolymorphisms, or (D) the computer system of any of (A) to (C), whereinthe computer system further comprises an identifier that identifies oneor more features in said sequence. 119-121. (canceled) 122: A computerreadable medium having stored thereon a polypeptide sequence or anucleic acid sequence, wherein the polypeptide sequence comprises thepolypeptide of claim 61; a polypeptide encoded by the nucleic acid ofclaim
 1. 123: A method for identifying a feature in a sequencecomprising: (a) reading the sequence using a computer program whichidentifies one or more features in a sequence, wherein the sequencecomprises a polypeptide sequence or a nucleic acid sequence, wherein thepolypeptide sequence comprises the polypeptide of claim 61; apolypeptide encoded by the nucleic acid of claim 1; and (b) identifyingone or more features in the sequence with the computer program. 124: Amethod for comparing a first sequence to a second sequence comprising:(A) (a) reading the first sequence and the second sequence through useof a computer program which compares sequences, wherein the firstsequence comprises a polypeptide sequence or a nucleic acid sequence,wherein the polypeptide sequence comprises the polypeptide of claim 61or a polypeptide encoded by the nucleic acid of claim 1; and (b)determining differences between the first sequence and the secondsequence with the computer program, or (B) the computer system of (A),wherein the step of determining differences between the first sequenceand the second sequence further comprises the step of identifyingpolymorphisms, or (C) the computer system of (A) or (B), wherein themethod further comprises an identifier that identifies one or morefeatures in a sequence, or (D) the computer system of any of (A) to (C),wherein the method further comprises reading the first sequence using acomputer program and identifying one or more features in the sequence.125-127. (canceled) 128: A method for isolating or recovering a nucleicacid encoding a polypeptide with a phospholipase activity from a samplecomprising the steps of: (A) (a) providing the amplification primersequence pair of claim 31; (b) isolating a nucleic acid from the sampleor treating the sample such that nucleic acid in the sample isaccessible for hybridization to the amplification primer pair; and, (c)combining the nucleic acid of step (b) with the amplification primerpair of step (a) and amplifying nucleic acid from the sample, therebyisolating or recovering a nucleic acid encoding a polypeptide with aphospholipase activity from the sample; or, (B) (a) providing apolynucleotide probe comprising the sequence of claim 1; (b) isolating anucleic acid from the sample or treating the sample such that nucleicacid in the sample is accessible for hybridization to a polynucleotideprobe of step (a); (c) combining the isolated nucleic acid or thetreated sample of step (b) with the polynucleotide probe of step (a);and (d) isolating a nucleic acid that specifically hybridizes with thepolynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide with a phospholipase activity fromthe sample, or (C) the method of (A) or (B), wherein the samplecomprises an environmental sample, or (D) the method of (C), wherein theenvironmental sample comprises a water sample, a liquid sample, a soilsample, an air sample or a biological sample, or (E) the method of (D),wherein the biological sample is derived from a bacterial cell, aprotozoan cell, an insect cell, a yeast cell, a plant cell, a fungalcell or a mammalian cell. 129-132. (canceled) 133: A method ofgenerating a variant of a nucleic acid encoding a polypeptide with aphospholipase activity comprising: (A) (a) providing a template nucleicacid comprising a sequence as set forth in claim 1; and (b) modifying,deleting or adding one or more nucleotides in the template sequence, ora combination thereof, to generate a variant of the template nucleicacid, or (B) the method of (A), wherein the method further comprisesexpressing the variant nucleic acid to generate a variant phospholipasepolypeptide, or (C) the method of (A) or (B), wherein the modifications,additions or deletions are introduced by a method comprising error-pronePCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,recursive ensemble mutagenesis, exponential ensemble mutagenesis,site-specific mutagenesis, gene reassembly, Gene Site SaturationMutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination,recursive sequence recombination, phosphothioate-modified DNAmutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation or acombination thereof, or (D) the method of and of (A) to (C), wherein themethod is iteratively repeated until a variant phospholipase having analtered or different activity or an altered or different stability fromthat of a polypeptide encoded by the template nucleic acid is produced,or (E) the method of (D), wherein the variant phospholipase polypeptideis thermotolerant, and retains some activity after being exposed to anelevated temperature, or (F) the method of (D), wherein the variantphospholipase polypeptide has increased glycosylation as compared to thephospholipase encoded by a template nucleic acid, or (G) the method of(D), wherein the variant phospholipase polypeptide has a phospholipaseactivity under a high temperature, wherein the phospholipase encoded bythe template nucleic acid is not active under the high temperature, or(H) the method of (A), wherein the method is iteratively repeated untila phospholipase coding sequence having an altered codon usage from thatof the template nucleic acid is produced, or (I) the method of (A),wherein the method is iteratively repeated until a phospholipase genehaving higher or lower level of message expression or stability fromthat of the template nucleic acid is produced. 134-142. (canceled) 143:A method for modifying codons in a nucleic acid encoding a polypeptidewith a phospholipase activity to increase its expression in a host cell,the method comprising: (A) (a) providing a nucleic acid encoding apolypeptide with a phospholipase activity comprising the nucleic acid ofclaim 1; and, (b) identifying a non-preferred or a less preferred codonin the nucleic acid of step (a) and replacing it with a preferred orneutrally used codon encoding the same amino acid as the replaced codon,wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell; or, (B) (a) providing a nucleic acidencoding a polypeptide with a phospholipase activity comprising thesequence of claim 1; and, (b) identifying a codon in the nucleic acid ofstep (a) and replacing it with a different codon encoding the same aminoacid as the replaced codon, thereby modifying codons in a nucleic acidencoding a phospholipase, or (C) the method of (A) or (B), wherein thehost cell is a bacterial cell, a fungal cell, an insect cell, a yeastcell, a plant cell or a mammalian cell. 144-147. (canceled) 148: Amethod for producing a library of nucleic acids encoding a plurality ofmodified phospholipase active sites or substrate binding sites, whereinthe modified active sites or substrate binding sites are derived from afirst nucleic acid comprising a sequence encoding a first active site ora first substrate binding site the method comprising: (A) (a) providinga first nucleic acid encoding a first active site or first substratebinding site, wherein the first nucleic acid sequence comprises asequence that hybridizes under stringent conditions to a sequence thatencodes a polypeptide having a sequence as set forth in SEQ ID NO:2; SEQID NO:175, or SEQ ID NO:176, or (b) SEQ ID NO:1, and the nucleic acidencodes a phospholipase active site or a phospholipase substrate bindingsite; (b) providing a set of mutagenic oligonucleotides that encodenaturally-occurring amino acid variants at a plurality of targetedcodons in the first nucleic acid; and, (c) using the set of mutagenicoligonucleotides to generate a set of active site-encoding or substratebinding site-encoding variant nucleic acids encoding a range of aminoacid variations at each amino acid codon that was mutagenized, therebyproducing a library of nucleic acids encoding a plurality of modifiedphospholipase active sites or substrate binding sites, or (B) the methodof (A), wherein the method further comprises mutagenizing the firstnucleic acid of step (a) by a method comprising an optimized directedevolution system, Gene Site Saturation Mutagenesis (GSSM), a syntheticligation reassembly (SLR), error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, recombination, recursive sequencerecombination, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesis,chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation, or a combination thereof. 149-163. (canceled) 164: Anisolated, synthetic or recombinant signal sequence consisting of asequence as set forth in residues 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1to 28, 1 to 28, 1 to 30 or 1 to 31, 1 to 32 or 1 to 33 of SEQ ID NO:2 orthe sequence of claim
 61. 165: A chimeric polypeptide comprising (a) atleast a first domain comprising signal peptide (SP) having a sequence asset forth in claim 164, and at least a second domain comprising aheterologous polypeptide or peptide, wherein the heterologouspolypeptide or peptide is not naturally associated with the signalpeptide (SP), or (b) the chimeric polypeptide of (a), wherein theheterologous polypeptide or peptide is not a phospholipase. 166.(canceled) 167: An isolated, synthetic or recombinant nucleic acidencoding a chimeric polypeptide, wherein the chimeric polypeptidecomprises at least a first domain comprising signal peptide (SP having asequence as set forth in claim 164 and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP). 168: A method of increasing thermotolerance orthermostability of a phospholipase polypeptide, the method comprisingglycosylating a phospholipase, wherein the polypeptide comprises atleast thirty contiguous amino acids of the polypeptide of claim 61, or apolypeptide encoded by the nucleic acid of claim 1, thereby increasingthe thermotolerance or thermostability of the phospholipase. 169: Amethod for overexpressing a recombinant phospholipase in a cellcomprising expressing a vector comprising the nucleic acid of claim 1,wherein overexpression is effected by use of a high activity promoter, adicistronic vector or by gene amplification of the vector. 170-174.(canceled) 175: A method for hydrolyzing, breaking up or disrupting aphospholipid-comprising composition comprising: (A) (a) providing thepolypeptide having a phospholipase activity of claim 61, or apolypeptide encoded by the nucleic acid of claim 1; (b) providing acomposition comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the phospholipase hydrolyzes, breaks up or disruptsthe phospholipid-comprising composition, or (B) the method of (A),wherein the composition comprises a phospholipid-comprising lipidbilayer or membrane, and optionally the composition comprises a plantcell, a bacterial cell, a yeast cell, an insect cell, or an animal cell,or (C) the method of (A), wherein the method comprises use of high shearmixing of the composition, followed by no or low shear mixing with theat least one polypeptide of the invention having a phospholipaseactivity to allow adequate contacting of the phospholipid substrate withthe phospholipase. 176-177. (canceled) 178: A method for liquefying orremoving a phospholipid-comprising composition comprising: (a) providingthe polypeptide having a phospholipase activity of claim 61, or apolypeptide encoded by the nucleic acid of claim 1; (b) providing acomposition comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the phospholipase removes or liquefies thephospholipid-comprising composition. 179: A detergent compositioncomprising (a) the polypeptide of claim 61, or a polypeptide encoded bythe nucleic acid of claim 1, wherein the polypeptide has a phospholipaseactivity, or (b) the detergent of (a), wherein the phospholipase is anonsurface-active phospholipase or a surface-active phospholipase, orthe phospholipase is formulated in a non-aqueous liquid composition, acast solid, a lyophilized powder, a granular form, a particulate form, acompressed tablet, a pellet, a gel form, a paste, an aerosol, or aslurry form. 180-181. (canceled) 182: A method for washing an objectcomprising: (a) providing a composition comprising the polypeptidehaving a phospholipase activity of claim 61, or a polypeptide encoded bythe nucleic acid of claim 1; (b) providing an object; and (c) contactingthe polypeptide of step (a) and the object of step (b) under conditionswherein the composition can wash the object. 183: A method for degummingan oil comprising: (A) (a) providing a composition comprising thepolypeptide having a phospholipase activity of claim 61, or apolypeptide encoded by the nucleic acid of claim 1; (b) providing ancomposition comprising an phospholipid-containing fat or oil; and (c)contacting the polypeptide of step (a) and the composition of step (b)under conditions wherein the polypeptide can catalyze the hydrolysis ofa phospholipid in the composition, or (B) the method of (A), wherein theoil-comprising composition comprises a plant, an animal, an algae or afish oil or fat, and optionally the plant oil comprises a rice bran oil,a soybean oil, a rapeseed oil, a corn oil, an oil from a palm kernel, acanola oil, a sunflower oil, a sesame oil or a peanut oil, or (C) themethod of (A) or (B), wherein the polypeptide hydrolyzes a phosphatidefrom a hydratable and/or a non-hydratable phospholipid in theoil-comprising composition, or optionally the polypeptide hydrolyzes aphosphatide at a glyceryl phosphoester bond to generate a diglycerideand water-soluble phosphate compound, or (D) the method of any of (A) to(C), wherein the polypeptide has a phospholipase C activity, oroptionally the polypeptide has a phospholipase D activity and aphosphatase enzyme is also added, and optionally the polypeptide havinga phospholipase activity has a PLC activity and the method increasesneutral oils, and optionally the polypeptide having a phospholipaseactivity has a PLC activity and the method increases diacylglycerol(DAG) production to contribute to an oil phase, or (E) the method of anyof (A) to (D), wherein the contacting comprises hydrolysis of a hydratedphospholipid in an oil, or (F) the method of any of (A) to (E), whereinthe hydrolysis conditions of step (c) comprise a temperature of about20° C. to 40° C. at an alkaline pH, or optionally the alkalineconditions comprise a pH of about pH 8 to pH 10, or (G) the method ofany of (A) to (F), wherein the hydrolysis conditions comprise analkaline pH, or the hydrolysis conditions of step (c) comprise areaction time of about 3 to 10 minutes, or the hydrolysis conditions ofstep (c) comprise hydrolysis of hydratable and non-hydratablephospholipids in oil at a temperature of about 50° C. to 60° C., at a pHof about pH 5 to pH 6.5, at a pH of about pH 6.0 to pH 7.5, or at a pHof about pH 5 to pH 8.0, using a reaction time of about 30 to 60minutes, or the alkaline conditions are sufficient to causeisomerization of a 1,2-DAG produced by a PLC into a 1,3-DAG, or (H) themethod of any of (A) to (G), wherein the polypeptide is bound to afilter and the phospholipid-containing fat or oil is passed through thefilter, or optionally the polypeptide is added to a solution comprisingthe phospholipid-containing fat or oil and then the solution is passedthrough a filter, or (I) the method of (A) or (H), wherein the methodfurther comprises addition of one or more polypeptides having aprotease, an amylase, a lipase, a cutinase, another phospholipase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase, or polypeptides with equivalent activity, or a combinationthereof, to further break down gum mass and enhance oil yields, or (J)the method of any of (A) to (I), wherein the method further comprisesphysical removal of gum produced by the degumming method by addition ofa hardening substance, or (K) the method of (J), wherein the finaldegummed oil product is enriched in 1,3-DAG, or (L) the method of any of(K), wherein the final degummed oil product comprises not less than 1.0%1,3-DAG. 184-196. (canceled) 197: A method for converting anon-hydratable phospholipid to a hydratable form comprising: (A) (a)providing a composition comprising the polypeptide having aphospholipase activity of claim 61, or a polypeptide encoded by thenucleic acid of claim 1; (b) providing an composition comprising anon-hydratable phospholipid; and (c) contacting the polypeptide of step(a) and the composition of step (b) under conditions wherein thepolypeptide converts the non-hydratable phospholipid to a hydratableform, or (B) the method of (A), wherein the polypeptide has aphospholipase C activity, or optionally the polypeptide has aphospholipase D activity and a phosphatase enzyme is also added.198-199. (canceled) 200: A method for caustic refining of aphospholipid-containing composition comprising: (A) (a) providing acomposition comprising a polypeptide having a phospholipase activity,wherein the polypeptide has the sequence of claim 61, or comprises apolypeptide encoded by the nucleic acid of claim 1; (b) providing ancomposition comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) before, duringor after the caustic refining, or (B) the method of (A), wherein thepolypeptide has a phospholipase C activity, or (C) the method of (A) or(B), wherein the polypeptide having a phospholipase activity is addedbefore addition of acid or caustic, or the polypeptide having aphospholipase activity is added during caustic refining and varyinglevels of acid and caustic are added depending on levels of phosphorusand levels of free fatty acids, or the polypeptide having aphospholipase activity is added after caustic refining: in an intensemixer or retention mixer prior to separation; following a heating step;in a centrifuge; in a soapstock; in a washwater; or, during bleaching ordeodorizing steps, or (D) the method of (C), wherein the causticrefining conditions are generated by addition of a concentrated solutionof caustic, and optionally the concentrated solution of caustic is moreconcentrated than the industrial standard of 11%, and optionally theconcentrated solution of caustic is between about 12% and 50%concentrated, or (E) the method of any of (A) to (D), wherein thecomposition comprising the phospholipid comprises a plant, andoptionally the polypeptide is expressed transgenically in the plant, andoptionally the polypeptide having a phospholipase activity added duringcrushing of a seed or other plant part, or, the polypeptide having aphospholipase activity added following crushing or prior to refining, or(F) the method of any of (A) to (E), wherein the method comprises aprocess as set forth in FIG.
 13. 201-204. (canceled) 205: A method forpurification of a phytosterol or a triterpene comprising: (A) (a)providing a composition comprising the polypeptide having aphospholipase activity of claim 61, or a polypeptide encoded by thenucleic acid of claim 1; (b) providing an composition comprising aphytosterol or a triterpene; and (c) contacting the polypeptide of step(a) with the composition of step (b) under conditions wherein thepolypeptide can catalyze the hydrolysis of a phospholipid in thecomposition, or (B) the method of (A), wherein the polypeptide has aphospholipase C activity, or (C) the method of (A) or (B), wherein thephytosterol or a triterpene comprises a plant sterol, or (D) the methodof any of (A) to (C), wherein the plant sterol is derived from avegetable oil, or (E) the method of (D), wherein the vegetable oilcomprises a coconut oil, canola oil, cocoa butter oil, corn oil,cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, oilderived from a rice bran, safflower oil, sesame oil, soybean oil or asunflower oil, or (F) the method of any of (A) to (E), wherein themethod further comprises use of nonpolar solvents to quantitativelyextract free phytosterols and phytosteryl fatty-acid esters, or (G) themethod of any of (A) to (F), wherein the phytosterol or a triterpenecomprises a β-sitosterol, a campesterol, a stigmasterol, a stigmastanol,a β-sitostanol, a sitostanol, a desmosterol, a chalinasterol, aporiferasterol, a clionasterol or a brassicasterol. 206-211. (canceled)212: A method for refining a crude oil comprising: (A) (a) providing acomposition comprising the polypeptide having a phospholipase activityof claim 61, or a polypeptide encoded by the nucleic acid of claim 1;(b) providing a composition comprising an oil comprising a phospholipid;and (c) contacting the polypeptide of step (a) with the composition ofstep (b) under conditions wherein the polypeptide can catalyze thehydrolysis of a phospholipid in the composition, or (B) the method of(A), wherein the polypeptide has a phospholipase C activity, or (C) themethod of (A) or (B), wherein the polypeptide having a phospholipaseactivity is in a water solution that is added to the composition, or thewater level is between about 0.5 to 5%, or the process time is less thanabout 2 hours, or the process time is less than about 60 minutes, or theprocess time is less than about 30 minutes, less than about 15 minutes,or less than about 5 minutes, or (D) the method of any of (A) to (C),wherein the hydrolysis conditions comprise a temperature of betweenabout 25° C.-70° C., or the hydrolysis conditions comprise use ofcaustics, or the hydrolysis conditions comprise a pH of between about pH3 and pH 10, or the hydrolysis conditions comprise addition ofemulsifiers and/or mixing after the contacting of step (c), or (E) themethod of any of (A) to (D), wherein the method further comprisesaddition of an emulsion-breaker and/or heat or cooling to promoteseparation of an aqueous phase, or (F) the method of any of (A) to (E),wherein the method further comprises degumming before the contactingstep to collect lecithin by centrifugation and then adding a PLC, a PLCand/or a PLA to remove non-hydratable phospholipids, or (G) the methodof any of (A) to (F), wherein the method further comprises waterdegumming of crude oil to less than 10 ppm phosphorus for edible oilsand subsequent physical refining to less than about 50 ppm phosphorusfor biodiesel oils, or (H) the method of any of (A) to (G), wherein themethod further comprises addition of acid to promote hydration ofnon-hydratable phospholipids, or (I) the method of any of (A) to (I),wherein sufficient acid is added to promote lowering of the calcium andmagnesium metal content. 213-226. (canceled) 227: A method for degummingan oil or a fat comprising: (a) providing a composition comprising thepolypeptide having a phospholipase activity of claim 61, or apolypeptide encoded by the nucleic acid of claim 1, wherein thephospholipase activity comprises a phospholipase D activity, and aphosphatase enzyme; (b) providing an composition comprising anphospholipid-containing fat or oil; and (c) contacting the polypeptideof step (a) and the composition of step (b) under conditions wherein thepolypeptide can catalyze the hydrolysis of a phospholipid in thecomposition. 228: A composition having the equivalent of a phospholipaseC activity comprising providing a composition comprising the polypeptidehaving a phospholipase activity of claim 61, or a polypeptide encoded bythe nucleic acid of claim 1, wherein the phospholipase activitycomprises a phospholipase D activity, and a phosphatase enzyme. 229: Amethod for ameliorating or preventing lipopolysaccharide (LPS)-mediatedtoxicity comprising administering to a patient a pharmaceuticalcomposition comprising the polypeptide of claim 61, or a polypeptideencoded by the nucleic acid of claim
 1. 230: A method for detoxifying anendotoxin comprising contacting the endotoxin with the polypeptide ofclaim 61, or a polypeptide encoded by the nucleic acid of claim
 1. 231:A method for deacylating a 2′ or a 3′ fatty acid chain from a lipid Acomprising contacting (A) the lipid A with the polypeptide of claim 61,or a polypeptide encoded by the nucleic acid of claim 1, or (B) themethod of (A), wherein the polypeptide has a patatin activity, or (C)the method of (B), wherein the patatin has a sequence as set forth inSEQ ID NO:12 (encoded by SEQ ID NO:11), SEQ ID NO:14 (encoded by SEQ IDNO:13), SEQ ID NO:18 (encoded by SEQ ID NO:17), SEQ ID NO:26 (encoded bySEQ ID NO:25), SEQ ID NO:28 (encoded by SEQ ID NO:27), SEQ ID NO:34(encoded by SEQ ID NO:33), SEQ ID NO:36 (encoded by SEQ ID NO:35), SEQID NO:44 (encoded by SEQ ID NO:43), SEQ ID NO:46 (encoded by SEQ IDNO:45), SEQ ID NO:56 (encoded by SEQ ID NO:55), SEQ ID NO:60 (encoded bySEQ ID NO:59), SEQ ID NO:66 (encoded by SEQ ID NO:65), SEQ ID NO:72(encoded by SEQ ID NO:71), SEQ ID NO:78 (encoded by SEQ ID NO:77), SEQID NO:87 (encoded by SEQ ID NO:86), SEQ ID NO:88 (encoded by SEQ IDNO:87), SEQ ID NO:92 (encoded by SEQ ID NO:91), SEQ ID NO:96 (encoded bySEQ ID NO:95), SEQ ID NO:100 (encoded by SEQ ID NO:99), SEQ ID NO:104(encoded by SEQ ID NO:103), SEQ ID NO:126 (encoded by SEQ ID NO:125),SEQ ID NO:128 (encoded by SEQ ID NO:127), SEQ ID NO:132 (encoded by SEQID NO:131), SEQ ID NO:134 (encoded by SEQ ID NO:133), SEQ ID NO:136(encoded by SEQ ID NO:135), or SEQ ID NO:138 (encoded by SEQ ID NO:137).232-250. (canceled) 251: A process for reducing gum mass and increasingneutral oil (triglyceride) gain through reduced oil entrapmentcomprising: (A) (a) providing a composition comprising the polypeptidehaving a phospholipase activity of claim 61, or a polypeptide encoded bythe nucleic acid of claim 1; (b) providing an composition comprising anphospholipid-containing fat or oil; and (c) contacting the polypeptideof step (a) and the composition of step (b) under conditions wherein thepolypeptide can catalyze the hydrolysis of a phospholipid in thecomposition for a time sufficient to reduce gum mass and increaseneutral oils, or (B) the method of (A), wherein the protein preparationcomprises a formulation comprising a non-aqueous liquid composition, acast solid, a powder, a lyophilized powder, a granular form, aparticulate form, a compressed tablet, a pellet, a pill, a gel form, ahydrogel, a paste, an aerosol, a spray, a lotion, a slurry formulation,an aqueous/oil emulsion, a cream, a capsule, a vesicle, or a micellarsuspension. 252-262. (canceled) 263: A method for making a variantphospholipase coding sequence having increased expression in a host cellcomprising modifying the nucleic acid sequence of claim 1 such that one,several or all N-linked glycosylation site motifs in the phospholipaseare modified to a non-glycosylated motif in the variant phospholipase.264. (canceled) 265: A method for making a variant phospholipase codingsequence encoding a phospholipase having increased resistance to aprotease comprising modifying position 131 of SEQ ID NO:2, or an aminoacid equivalent to position 131 in the amino acid sequence of claim 61,2 to one, several or all of the following residues: Lysine (K); Serine(S); Glycine (G); Arginine (R); Glutamine (Q); Alanine (A); Isoleucine(I); Histidine (H); Phenylalanine (F); Threonine (T); Methionine (M)Leucine (L).
 266. (canceled) 267: A method for making a variantphospholipase coding sequence encoding a phospholipase having decreasedresistance to a protease comprising modifying position 131 of SEQ IDNO:2, or an amino acid equivalent to position 131 in the amino acidsequence of claim 61, 2 to one, several or all of the followingresidues: Tryptophan (W); Glutamate (E); Tyrosine (Y).
 268. (canceled)269: A method for making and expressing a protein having a biologicalactivity whose activity is temporarily inactivated by glycosylationcomprising: (A) (a) providing a nucleic acid encoding a protein having abiological activity, wherein the protein is not naturally glycosylated,and the polypeptide comprises the polypeptide of claim 61, or comprisesa polypeptide encoded by the nucleic acid of claim 1; (b) inserting atleast one glycosylation motif coding sequence into the protein-encodingnucleic acid, wherein the glycosylated form of the protein is inactive;(c) inserting a targeting sequence into the protein such that it isdirected to a host cell's secretory pathway, wherein the host cell iscapable of recognizing the glycosylation motif and glycosylating theprotein; and (d) expressing the modified nucleic acid in the host cell,or (B) the method of (A), wherein the method further comprisesdeglycosylating the expressed the protein, thereby re-activating theactivity of the protein, or (C) the method of (A) or (B), wherein thehost cell is a eukaryotic cell. 270-271. (canceled) 272: A method forexpressing phospholipase C comprising (A) (a) providing a Pichia strainwith a Mut+phenotype; (b) inserting a heterologous phospholipaseC-encoding nucleic acid in the Pichia strain, wherein the phospholipaseC-encoding nucleic acid comprises the nucleic acid of claim 1; and, (c)culturing the Pichia strain under conditions whereby the phospholipase Cis expressed, or (B) the method of (A), wherein the method furthercomprises supplementing the culture conditions with zinc. 273.(canceled) 274: A cell system for expressing phospholipase C comprisinga Mut+phenotype Pichia strain comprising a heterologous phospholipaseC-encoding nucleic acid operably linked to a promoter operable in thePichia strain, wherein the phospholipase C-encoding nucleic acidcomprises the nucleic acid of claim
 1. 275: A cell system for expressinga heterologous protein comprising a Pichia strain cell resistant tozeocin, wherein the heterologous protein is encoded by a nucleic acidcomprising the nucleic acid of claim 1, or the heterologous proteincomprises the polypeptide of claim
 61. 276: A zeocin-resistant yeastcell system for expressing a heterologous protein comprising the stepsof (A) (a) providing a Pichia sp. cell comprising a heterologous nucleicacid capable of expressing a heterologous protein, wherein theheterologous protein-encoding nucleic acid comprises the nucleic acid ofclaim 1; (b) culturing the cell under conditions comprising zeocin at aninitial concentration; (c) selecting cells resistant to the initialconcentration of zeocin, and reculturing under conditions comprising ahigher concentration of zeocin; and (d) selecting the cells cultured instep (c) resistant to the higher concentration of zeocin or (B) themethod of (A), wherein the heterologous protein is an enzyme, or (C) themethod of (B), wherein the enzyme is a phospholipase, or a phospholipaseC (PLC).
 277. (canceled)